cells, like the carboxysome no discernable stages of polyhedra assembly were noted during the time course (Orus et al. 1995).

Screening of TPOP Mutants

      A pool of TPOP insertions was tested for their ability to produce acid and aldehydes and the ability to use PD as a sole carbon and energy source using MacConkey/B12/PD, AIM, and PD minimal media plates, respectively. TPOP insertions are polar in the absence of tetracycline, however, when tetracycline is present expression out of both ends of the insertion is induced effectively making the insertion nonpolar (Way et al. 1984, Rappleye and Roth 1997). Five mutants were selected for further study to determine the effects of the TPOP insertions on polyhedral organelle production. The results of the study are represented in Table 2-3. As expected, strains BE34 and BE35, which both carry TPOP insertions in the pduC gene, encoding the large subunit of diol dehydratase, were unable to use PD for growth; however, it was surprising that strains BE33 and BE37, which carry TPOP insertions in the pduA gene, and strain BE38, which carries an insertion in the pduB gene, were either unable to or exhibited a reduced ability to use PD for growth. Both thepduA andpduB genes encode proteins homologous to those required for carboxysome formation, and these strains were of particular interest since their inability to synthesize the polyhedral organelles suggests an effect on growth using PD.

Effect of TPOP Insertions on the Formation of Polyhedral Organelles

      The ability of the five insertion mutants to form polyhedral organelles was

examined (Table 2-3). Strains BE33-BE37 containing TPOP insertion mutations were grown on succinate/PD minimal medium containing tetracycline to induce expression of genes downstream of the TPOP insertion. Cells were grown to late log phase, harvested,