Molecular interactions involved in the biogenesis of bacterial microcompartments

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Chemistry and Biochemistry

First Advisor

Gordon C. Cannon

Advisor Department

Chemistry and Biochemistry


This study was undertaken with the goal of gaining better insights into the assembly pathway of carboxysomes and related polyhedra. Aside from their similarity in size and shape, all known microcompartments package enzymes that mediate key reactions of metabolic pathways [1]. It remains unclear whether the sequestered enzymes participate in microcompartment assembly and/or contribute to the overall shape of the polyhedra. Genetic studies in Salmonella enterica have suggested that the shells of organelles involved in propanediol utilization assemble in the absence of encapsulated enzymes [2]. In this study, in vivo yeast two-hybrid screens, involving components of the e[barbelow]thanolamine u[barbelow]tilization (Eut) organelles of S. enterica , revealed strong interactions between the putative shell proteins--EutN and EutL [3, 4], and the large subunit of the ethanolamine ammonia lyase enzyme. This result suggested that similar interactions between shell components and sequestered enzymes may play an important role in the biogenesis of other microcompartments as well. Since the Eut polyhedra could not be purified and characterized, conclusions drawn from the yeast two-hybrid protein interaction studies remain speculative. A more direct in vivo approach for addressing the role of packaged enzymes in microcompartment assembly was employed by constructing Form IA RubisCO mutants of the model chemoautotrophic bacterium, Halothiobacillus neapolitanus , and analyzing their phenotypes. In these mutants, the genes encoding Form IA RubisCO, the enzyme sequestered within carboxysomes, were either deleted or replaced with orthologs from another autotrophic bacterium, Thiomicrospira crunogena. Phenotypic characterization studies revealed that a mutant lacking Form IA RubisCO assembled empty carboxysome shells of apparently normal size, shape, and composition. Furthermore, carboxysomes of H. neapolitanus readily packaged chimeric and heterologous species of Form IA RubisCO. The large subunit of these foreign RubisCO species was identified as an important determinant of the enzyme's packagability. The impact of these findings on the current understanding of carboxysome architecture and function will be discussed.