Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement

Abstract

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

The effects of time, temperature, and pH on the stability of PDU bacterial microcompartments

“…While these may still be intact MCPs, another potential explanation is that the MCPs and the reporter protein formed aggregates. In agreement with this latter hypothesis, a time course of cells expressing PduP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] -GFP in the absence of MCP induction shows diffuse fluorescence for several days, and also shows potential aggregation after eight days, as seen by the emergence of fluorescent puncta (Supporting Information 2, Fig. S1).…”

“…1(B)]. [7][8][9][10] The structures of several of these shell proteins have been solved, providing insight on how the subunits assemble to form the polyhedral shell. [11][12][13] The major constituents of the Pdu MCP shell form homohexamers and self-assemble to create the facets of the MCP [ Fig.…”

Cellular microcompartments constitute general suborganellar functional units in cells

“…their structure similar to a virus shell and, indeed, rely on similar very simple building blocks Parsons et al , 2010 ;Tanaka et al , 2010 ).…”

Modularity of a carbon-fixing protein organelle

“…1073/pnas.1108557109/-/DCSupplemental. compartments in Escherichia coli (22,23). In addition, a putative N-terminal signal sequence capable of loading new proteins into the PDU has been isolated; however, a targeting sequence has not yet been elucidated for CB proteins (20,24).…”