role for the pdu organelles in aldehyde detoxification and also show that these organelles represent a complex mode of subcellular organization.



                             Materials and Methods

Chemicals and Reagents

     PD was from Sigma Chemical Company (St. Louis, MO), Bacterial Protein

Extraction Reagent II (B-PER II) was from Pierce (Rockford, IL), and Pefabloc SC was from Pentapharm Ltd. (Basel, Switzerland). Electrophoresis supplies were from Bio-Rad (Hercules, CA) unless otherwise stated. Other chemicals were from Fisher Scientific (Pittsburgh, PA).

Organelle Purification

     For organelle purification, S. enterica serovar Typhimurium LT2 was grown in 2.8

1 Fernbach flasks containing 1 1 of NCE minimal medium (Vogel and Bonner 1956, Berkowitz et al. 1968) supplemented with 1% succinate and 0.4% PD. The inoculum was a 5 ml Luria-Bertani (LB) broth culture and incubation was at 37°C with shaking at 275 RPM. Under these conditions, growth was supported by succinate, but the PD was not metabolized assuring continued high induction of the pdu operon (Chen et al. 1995, Bobik et al. 1997). After cultures reached late log phase (OD600 between 1 and 1.2), cells from 2 1 of media were harvested by centrifugation at 4,000 x g forlO min. at room temperature. The pelleted cells (5-6 grams) were washed with 300 ml of lysozyme buffer (50 mM Tris-HCl, 0.6 M sucrose, 5 mM EDTA, 0.2% PD, pH 8.0), resuspended in 30 ml of similar buffer containing 2 mg/ml lysozyme, and incubated at 37°C for 2 hr with occasional agitation. Subsequent to digestion with lysozyme all steps were performed at 0-4°C. Cells were pelleted by centrifugation (7,740 x g, 15 min.), washed with lysozyme