the same restriction sites. The ligation reaction was used to transform E. coi S 17.1 Apir via electroporation, and one clone (plasmid pGH13) containing an insert of expected size (3,666 bp) was used to introduce the deletion into the S. enterica chromosome using the procedure of Miller and Mekelanos with some modification (Miller and Mekalanos 1988). For the conjugation step, BE47 was used as the recipient, and Ampr and Camr colonies were selected. Instead of the sucrose sensitivity screen used in the protocol, MacConkey/PD/CN-B12 plates were used to screen for colonies that had lost the ability to degrade PD. Deletion strains were identified by PCR using whole cells as a source of template. A transducing phage lysate of LT2 was used to cross off the Cam marker in strain BE79 to make strain BE87.

                                     Results

Polyhedral Organelles are Formed in Cells Grown on Poor Carbon Sources in the Presence of PD

      In order to define the conditions necessary for polyhedral organelle formation in S. enterica, wild type cells were grown to late log phase in liquid media using various carbon sources under different environmental conditions and then examined for the presence of polyhedral organelles using electron microscopy. The results of the tests are displayed in Table 2-2. When grown on PD, S. enterica formed polyhedral organelles (Figure 2-2A). This was observed during both aerobic growth on PD and during anaerobic growth on PD when tetrathionate was supplied as the terminal electron acceptor. Tetrathionate was used as an electron acceptor for respiration of PD since the more common electron acceptors nitrate, fumarate, trimethaline-N-oxide (TMAO) or dimethyl sulfoxide (DMSO) do not support growth on PD (Price-Carter et al. 2001). The only other growth conditions under which polyhedral organelle formation was observed