level) resulted in no observable difference in polyhedral organelle formation indicating that anaerobic respiration using tetrathionate as an electron acceptor is not required for polyhedral organelle formation. Interestingly, only rarely were polyhedral organelles observed in cells grown anaerobically on glycerol using either tetrathionate or fumarate as an electron acceptor. These conditions have been demonstrated previously to highly induce the pdu operon and the reduction of organelle formation suggests that PD may play some role in the formation of the polyhedral organelles in addition to its role in induction of thepdu operon.

Time Course of Organelle Formation

      The time course of polyhedral organelle formation was followed during aerobic growth of S enterica on PD minimal medium (Figure 2-3). Samples were taken during different phases of growth and observed using electron microscopy. The polyhedral organelles proved difficult to discern from other cytoplasmic constituents even with the utilization of improved staining techniques designed to impart extra contrast to the polyhedral organelles (Bobik et al. 1999). To facilitate the identification of polyhedral organelles, cells were labeled with a polyclonal antiserum generated against diol dehydratase, an enzyme formerly shown to be associated with the polyhedral organelles (Bobik et al. 1999). As expected, the polyhedral organelles were not observed in the LB inoculum. After transfer to PD minimal medium, polyhedral organelles were observed in cells as early as 5 hr subsequent to the transfer. The number of polyhedral organelles was highest at this point, remained constant throughout the log phase and seemed to decrease in the remainder of the growth curve. Nevertheless, polyhedral organelles were observed in cells throughout the remainder of the growth curve and were present even in dead