Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis

Abstract

Many heterotrophic bacteria have the ability to make polyhedral structures containing metabolic enzymes that are bounded by a unilamellar protein shell (metabolosomes or enterosomes). These bacterial organelles contain enzymes associated with a specific metabolic process (e.g. 1,2-propanediol or ethanolamine utilization). We show that the 21 gene regulon specifying the pdu organelle and propanediol utilization enzymes from Citrobacter freundii is fully functional when cloned in Escherichia coli, both producing metabolosomes and allowing propanediol utilization. Genetic manipulation of the level of specific shell proteins resulted in the formation of aberrantly shaped metabolosomes, providing evidence for their involvement as delimiting entities in the organelle. This is the first demonstration of complete recombinant metabolosome activity transferred in a single step and supports phylogenetic evidence that the pdu genes are readily horizontally transmissible. One of the predicted shell proteins (PduT) was found to have a novel Fe-S center formed between four protein subunits. The recombinant model will facilitate future experiments establishing the structure and assembly of these multiprotein assemblages and their fate when the specific metabolic function is no longer required.It has been recognized for more than 30 years that all cyanobacteria (1) and some other chemoautotrophic bacteria (2) contain carboxysomes. These polyhedral cellular inclusions consist of a proteinaceous shell enclosing an active enzyme, ribulose bisphosphate carboxylase/oxygenase (RuBisCO). 4Their function is to enhance the fixation of carbon dioxide (3), a reaction of planetary significance in that marine cyanobacteria are responsible for the majority of global carbon fixation (4, 5). More recently, sequence similarity was noticed between carboxysome shell genes and metabolic operon genes associated with propanediol utilization (pdu) and ethanolamine utilization (eut) in a variety of heterotrophic bacteria found in the mammalian gut (3) and the environment. In growth conditions that induce these metabolic operons, polyhedral organelles resembling carboxysomes were observed on electron microscopy of Salmonella enterica serovar Typhimurium (6), Klebsiella oxytoca, Citrobacter freundii, and Escherichia coli (7). Bioinformatics analysis also locates genes resembling carboxysome shell genes in metabolic operons in Clostridium perfringens (8), Clostridium tetani (9), Listeria monocytogenes and Listeria innocua (10), Enterococcus faecalis (11), Lactobacillus collinoides (12), Citrobacter rodentium, 5 and Yersinia enterocolitica (13) among other organisms. The non-carboxysome polyhedral structures have been referred to as enterosomes (3) or metabolosomes (14), emphasizing their role in cellular metabolism.There is some considerable interest in how these proteinaceous organelles form and the arrangement of protein subunits that give rise to these remarkable macromolecular assemblies. In carboxysomes, there are thought to be a number of shell prot...

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.…”

Short N-terminal sequences package proteins into bacterial microcompartments

“…With regard to biotechnology, it has been suggested that bacterial MCPs might find application as specialized reaction chambers for the production of industrial chemicals (13,(43)(44)(45)(46)(47). A major challenge in engineering pathways for the production of biofuels, green chemicals, and pharmaceuticals is balancing enzyme activities to limit the accumulation of pathway intermediates, particularly those that may be toxic to host cells.…”

Bacterial Microcompartments

“…The lateral transfer of genetic modules encoding BMCs appears to be a frequent mechanism for adding to the core repertoire of bacterial genomes to expand the metabolic capacity of the recipient and enable adaptation to new ecological niches. As such, BMCs have tremendous potential for bioengineering of metabolic modules in bacteria; the first proof-of-concept for this approach was recently described by Parsons et al (46).…”