Key points are not available for this paper at this time.
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. 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 (1Shively J.M. Ball F. Brown D.H. Saunders R.E. Science. 1973; 182: 584-586Crossref PubMed Scopus (220) Google Scholar) and some other chemoautotrophic bacteria (2Shively J.M. van Keulen G. Meijer W.G. Annu. Rev. Microbiol. 1998; 52: 191-230Crossref PubMed Scopus (227) Google Scholar) contain carboxysomes. These polyhedral cellular inclusions consist of a proteinaceous shell enclosing an active enzyme, ribulose bisphosphate carboxylase/oxygenase (RuBisCO). 4The abbreviations used are: RuBisCO, ribulose bisphosphate carboxylase/oxygenase; FFT, fast Fourier transformation; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. Their function is to enhance the fixation of carbon dioxide (3Cannon G.C. Bradburne C.E. Aldrich H.C. Baker S.H. Heinhorst S. Shively J.M. Appl. Environ. Microbiol. 2001; 67: 5351-5361Crossref PubMed Scopus (182) Google Scholar), a reaction of planetary significance in that marine cyanobacteria are responsible for the majority of global carbon fixation (4Shively J.M. English R.S. Baker S.H. Cannon G.C. Curr. Opin. Microbiol. 2001; 4: 301-306Crossref PubMed Scopus (43) Google Scholar, 5Heffelfinger G.S. Martino A. Gorin A. Xu Y. Rintoul III, M.D. Geist A. Al-Hashimi H.M. Davidson G.S. Faulon J.L. Frink L.J. Haaland D.M. Hart W.E. Jakobsson E. Lane T. Li M. Locascio P. Olken F. Olman V. Palenik B. Plimpton S.J. Roe D.C. Samatova N.F. Shah M. Shoshoni A. Strauss C.E. Thomas E.V. Timlin J.A. Xu D. OMICS. 2002; 6: 305-330Crossref PubMed Scopus (10) Google Scholar). 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 (3Cannon G.C. Bradburne C.E. Aldrich H.C. Baker S.H. Heinhorst S. Shively J.M. Appl. Environ. Microbiol. 2001; 67: 5351-5361Crossref PubMed Scopus (182) Google Scholar) 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 (6Bobik T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar), Klebsiella oxytoca, Citrobacter freundii, and Escherichia coli (7Havemann G.D. Bobik T.A. J. Bacteriol. 2003; 185: 5086-5095Crossref PubMed Scopus (127) Google Scholar). Bioinformatics analysis also locates genes resembling carboxysome shell genes in metabolic operons in Clostridium perfringens (8Shimizu T. Ohtani K. Hirakawa H. Ohshima K. Yamashita A. Shiba T. Ogasawara N. Hattori M. Kuhara S. Hayashi H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 996-1001Crossref PubMed Scopus (591) Google Scholar), Clostridium tetani (9Bruggemann H. Baumer S. Fricke W.F. Wiezer A. Liesegang H. Decker I. Herzberg C. Martinez-Arias R. Merkl R. Henne A. Gottschalk G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1316-1321Crossref PubMed Scopus (291) Google Scholar), Listeria monocytogenes and Listeria innocua (10Buchrieser C. Rusniok C. Kunst F. Cossart P. Glaser P. FEMS Immunol. Med. 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The non-carboxysome polyhedral structures have been referred to as enterosomes (3Cannon G.C. Bradburne C.E. Aldrich H.C. Baker S.H. Heinhorst S. Shively J.M. Appl. Environ. Microbiol. 2001; 67: 5351-5361Crossref PubMed Scopus (182) Google Scholar) or metabolosomes (14Brinsmade S.R. Paldon T. Escalante-Semerena J.C. J. Bacteriol. 2005; 187: 8039-8046Crossref PubMed Scopus (98) Google Scholar), 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 proteins that encase the RuBisCO and carbonic anhydrase. The situation is more complex in metabolosomes, where there are at least five shell proteins that encase ancillary factors, metabolic enzymes, and activating factors. There are thus between 17 and 21 genes associated with the ethanolamine and propanediol metabolosomes, respectively, and although some functional studies have been undertaken, little is known about the topological arrangement of the encoded protein components within the organelle. However, sequence analysis reveals that the shell proteins found in carboxysomes and metabolosomes are similar, indicating that they have evolved from a common ancestor. Structural studies on some of the individual shell proteins have given an insight into how they may function. The main carboxysome shell protein, CCMK1, has been shown to have a hexameric crystal structure with a charged pore (15Kerfeld C.A. Sawaya M.R. Tanaka S. Nguyen C.V. Phillips M. Beeby M. Yeates T.O. Science. 2005; 309: 936-938Crossref PubMed Scopus (328) Google Scholar), suggesting a selective permeability mechanism making the structure a prokaryotic functional equivalent to a eukaryotic organelle. Structures of the CsoS1A carboxysome protein from Halothiobacillus neapolitanus (16Tsai Y. Sawaya M.R. Cannon G.C. Cai F. Williams E.B. Heinhorst S. Kerfeld C.A. Yeates T.O. PLoS Biol. 2007; 5: e144Crossref PubMed Scopus (120) Google Scholar) and the EutN shell protein of the ethanolamine utilization enterosomes from E. coli (17Forouhar F. Kuzin A. Seetharaman J. Lee I. Zhou W. Abashidze M. Chen Y. Yong W. Janjua H. Fang Y. Wang D. Cunningham K. Xiao R. Acton E. L. J. 2007; PubMed Scopus Google Scholar) also hexameric with charged a microscopy of carboxysomes they have an with a complete was of a in individual carboxysome of in carboxysome and of on the F. Aldrich H.C. W. Shively J.M. J. Biol. PubMed Scopus Google Scholar). has suggesting that carboxysomes from a contain are into or four C.V. D.M. Martino A. J. Biol. 2007; PubMed Scopus Google Scholar). The and of both the genes for the protein shell (15Kerfeld C.A. Sawaya M.R. Tanaka S. Nguyen C.V. Phillips M. Beeby M. Yeates T.O. 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We for evidence of of E. coli on in the of 1,2-propanediol metabolosomes E. coli on in the of 1,2-propanediol show metabolosomes the on E. coli metabolosomes in the or of In on the metabolosomes were and formed to of the observed containing the structures when In the metabolosomes on their There were in the of with or of a polyhedral structure with and longer These are to for metabolosomes formed in S. enterica (7Havemann G.D. Bobik T.A. J. Bacteriol. 2003; 185: 5086-5095Crossref PubMed Scopus (127) Google Scholar), and carboxysomes from H. neapolitanus also show with F. Aldrich H.C. W. Shively J.M. J. Biol. 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It was also observed that an E. coli a with the main C. freundii pdu genes in and propanediol by electron microscopy of that no However, with a containing both and resulted in of activity and the formation of the of the metabolosome to enhance the metabolic activity of the as the and containing or was to form metabolosomes in E. coli that and are both for metabolosome the of both and on that both proteins are components of the formation is the of as little or of some of the individual proteins organelle formation or to the formation of of and and are all predicted to be shell in an E. coli that the pdu operon rise to The in and form of the organelle when these proteins are a role for these proteins in the shell of the organelle. The organelles were and no were to their protein with that of no protein studies have been to the shell proteins form the shell of the of of these that a with an within a indicating that the organelle has some form of The of a a by single electron or be organelle the or other in the shell This is an insight into the metabolic of the may also be by the It is to that the also an Fe-S center Bobik T.A. 2005; PubMed Scopus Google Scholar), P. and M. J. unpublished material. and thus and form of a to of into the organelle the of the It is also that was found to be associated with the The of the pdu regulon from C. freundii a on the as the of the from S. enterica into E. coli E. A. F. Warren M.J. E. A. C. J. Bacteriol. PubMed Scopus Google Scholar). has a to the role of individual components of the propanediol utilization There is to be about metabolosomes, as how the components are how the enzymes are and how and are in and of the organelle T.A. Appl. Microbiol. 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Parsons et al. (Tue,) studied this question.