recognizing the PduJ protein and E. coli proteins from the anti-PduA polyclonal antiserum preparation. After adsorption, the antiserum preparation recognized a singleband at 9.5 kDa in the wild type strain (Figure 3-3B, lane 1), and no labeling was observed in the pduA deletion mutant, BE182 (Figure 3-3B, lane 2). The absorbed polyclonal antiserum preparation also recognized a 9.5 kDa protein band in strain BE233 which overexpresses the PduA protein (Figure 3-3B, lane 4). No labeling at 9.5 kDa was observed in control strain BE232 which carried the plasmid without insert (Figure 3-3B, lane 3). These results showed that the absorbed anti-PduA antiserum preparation was highly specific for the PduA protein, and this antiserum preparation was used for subsequent immunolabeling experiments.

Localization of PduA by Immunoelectron Microscopy

      Immunogold labeling of S. enterica cells with highly specific adsorbed anti-PduA antiserum (see above) indicated that the PduA protein is a component of the shell of the polyhedral organelles. In the micrograph shown in Figure 3-4C, the antibody-conjugated gold particles (solid black circles) indicate the location of the PduA protein. The majority of the gold particles localized to the periphery of the polyhedral organelles. Preimmune serum failed to label the polyhedra (data not shown), and only a small amount of spurious labeling was observed when strain BE182 (ApduA) was labeled with anti-PduA antiserum (Figure 3-4D).

Localization of Diol Dehydratase in a pduA Mutant

      Labeling of wild type and BE182 (lpduA) cells with anti-diol dehydratase

antibodies illustrated the effect of a nonpolarpduA deletion mutation on the localization of diol dehydratase (Figure 3-4A and 3B). In the wild type strain, diol dehydratase