1987) with the following modifications: pararosaniline was added to sterile medium as a fine powder, 200 ng/ml of CN-B12 was included, and ethanol was replaced by 1% PD. TPOP Mutagenesis

     The Tnl0dTet elements used to create nonpolar insertion mutants in the strains used in this study are transposition defective derivatives of TnlO from which the transposase gene and the internal ends of ISIO have been deleted (Way et al. 1984). In addition, these transposons, referred to as TPOPs, have been further modified in order to increase transcription from the tetracycline inducible promoters (Rappleye and Roth 1997). Hence TPOPS are nonpolar in the presence of tetracycline but polar in its absence. For TPOP mutagenesis of thepdu region, a pool of approximately 80,000 independent insertion mutations was prepared as described in Bobik et al. (Bobik et al. 1992). The pool was then used as a donor in transductional crosses with strain BE235 (pdul2:.MudJ). Tetracycline resistance was selected, and transductants were screened for loss of the pdu]l2::.MudJ elements using MacConkey/lactose/PD indicator plates (Bobik et al. 1992). Following transposon mutagenesis, 30,000 colonies were screened, and 120 TPOP1 insertions located near thepdu operon were isolated. This work was performed by Lydia Jorge and Dr. Thomas Bobik. Localization of TPOP Insertions

     PCR was used to amplify the region of DNA that included one join-point between the TPOP element and the S. enterica chromosome. The primers used for amplification in strain BE33 were: forward, 5'ACCTTTGGTCACCAACGCTTTTCC-3' and reverse, 5'-GTTCATATGCGAAACCACTTC-3'. The DNA sequence of the PCR product was determined, and the location of the downstream join-point was determined to be bp 160 of the pduA coding sequence. The localization of the other TPOP insertions was