Biochemical and Structural Insights into Bacterial Organelle Form and Biogenesis
2008; Elsevier BV; Volume: 283; Issue: 21 Linguagem: Inglês
10.1074/jbc.m709214200
ISSN1083-351X
AutoresJoshua B. Parsons, Sriramulu Diraviam Dinesh, Evelyne Deery, Helen K. Leech, Amanda A. Brindley, Dana Heldt, Stefanie Frank, C. Mark Smales, Heinrich Lünsdorf, Alain Rambach, Mhairi Gass, Andrew Bleloch, Kirsty J. McClean, Andrew W. Munro, Stephen E. J. Rigby, Martin J. Warren, Michael B. Prentice,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoMany 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. Microbiol. 2003; 35: 207-213Crossref PubMed Scopus (166) Google Scholar), Enterococcus faecalis (11Paulsen I.T. Banerjei L. Myers G.S. Nelson K.E. Seshadri R. Read T.D. Fouts D.E. Eisen J.A. Gill S.R. Heidelberg J.F. Tettelin H. Dodson R.J. Umayam L. Brinkac L. Beanan M. Daugherty S. DeBoy R.T. Durkin S. Kolonay J. Madupu R. Nelson W. Vamathevan J. Tran B. Upton J. Hansen T. Shetty J. Khouri H. Utterback T. Radune D. Ketchum K.A. Dougherty B.A. Fraser C.M. Science. 2003; 299: 2071-2074Crossref PubMed Scopus (743) Google Scholar), Lactobacillus collinoides (12Sauvageot N. Muller C. Hartke A. Auffray Y. Laplace J.M. FEMS Microbiol. Lett. 2002; 209: 66-71Crossref Google Scholar), Citrobacter rodentium, 5M. Prentice, unpublished material. and Yersinia enterocolitica (13Prentice M.B. Cuccui J. Thomson N. Parkhill J. Deery E. Warren M.J. Adv. Exp. Med. Biol. 2003; 529: 43-46Crossref PubMed Google Scholar) among other organisms. 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 T.B. Pichersky E. Klessig D.F. Porter C.W. Montelione G.T. Tong L. J. Struct. Funct. Genomics. 2007; 8: 37-44Crossref PubMed Scopus (27) Google Scholar) also reveal similar hexameric arrangements with charged residues surrounding a central pore. Cryoelectron microscopy of extracted carboxysomes shows they have an icosahedral symmetry with triangular faces, but a complete image reconstruction was not possible because of a variability in individual carboxysome size, lack of symmetry in carboxysome contents, and absence of obvious capsomers on the surface (18Schmid M.F. Paredes A.M. Khant H.A. Soyer F. Aldrich H.C. Chiu W. Shively J.M. J. Mol. Biol. 2006; 364: 526-535Crossref PubMed Scopus (110) Google Scholar). A similar imaging project has provided even further detail, suggesting that carboxysomes isolated from a Synechococcus strain contain ∼250 RuBisCOs, which are organized into three or four concentric layers (19Iancu C.V. Ding H.J. Morris D.M. Dias D.P. Gonzales A.D. Martino A. Jensen G.J. J. Mol. Biol. 2007; 372: 764-773Crossref PubMed Scopus (116) Google Scholar). The distribution and relatedness of both the structural genes for the protein shell (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) and the associated metabolic operons (20Lawrence J.G. Roth J.R. Genetics. 1996; 142: 11-24Crossref PubMed Google Scholar, 21Porwollik S. Wong R.M. McClelland M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8956-8961Crossref PubMed Scopus (203) Google Scholar) strongly suggest repeated horizontal transmission in evolution. In keeping with this, we show that plasmid-based expression in E. coli of a pdu operon from C. freundii containing metabolosome structural and enzyme genes results in synthesis of recombinant metabolosomes with fully functional enzyme activity. In this way we have compartmentalized the bacterial cytoplasm, showing that it is possible to create novel compartments by horizontal genetic transfer in bacteria and to vary the shape and topology of the organelle by overproducing individual shell proteins. This process has the potential to be used for metabolic engineering whereby bacteria can be shielded from toxic metabolic intermediates. Thus, this project will contribute to the emerging discipline of synthetic biology. A more detailed account of the techniques, including the cloning and sequencing of the pdu operon is given in the supplemental material. Citrobacter sp. Library—The genomic DNA of Citrobacter sp. was partially digested with Sau3AI, and 20–30-kb fragments were purified after separation by agarose gel electrophoresis, cloned into the BglII site of the cosmid pLA 2917, and encapsidated in vitro following the Packagene® Lambda DNA packaging system instructions (Promega). The library was used to infect E. coli LE392. One cosmid, which had the ability to degrade 1,2-propanediol, was isolated and named pAR3114 (supplemental Table S1). Bacteria and Growth Conditions—All strains and plasmids are described in supplemental Table S1. E. coli JM109 wild-type and recombinant strains harboring pLA2917 (22Allen L.N. Hanson R.S. J. Bacteriol. 1985; 161: 955-962Crossref PubMed Google Scholar) and pAR3114 were grown either in LB medium or trypticase soy agar (10 g of tryptone, 5 g of NaCl, and 15 g of agar in 1 liter of distilled water) without or with tetracycline (20 μg/ml), respectively. pAR3114 is a pLA2917 cosmid containing an ∼30-kb (pdu operon, cbiA and pocR genes) insert derived from Sau3AI digestion of the C. freundi chromosome. Sequencing of Insert, Gene Annotation, and Recombinant Manipulation—The pdu operon sequence was obtained from pAR3114 after it was sent to be sequenced by Qiagen Ltd. The insert was found to contain 28,618 bp. The sequencing coverage was higher than 12, and the data quality has an error rate of 99.99%. The cloning and overproduction of individual Pdu components was achieved as described in the supplemental material. Propanediol Utilization Assay—The utilization of 1,2-propanediol was tested by a MacConkey triple-plate method (20Lawrence J.G. Roth J.R. Genetics. 1996; 142: 11-24Crossref PubMed Google Scholar). Bacteria were streaked on to MacConkey agar (20 g Soy peptone, 5 g of ox bile, 5 g of NaCl, 0.075 g of neutral red, 1% 1,2-propanediol, and 12 g of agar in 1 liter of distilled water) plates containing 1,2-propanediol, 20 μm cobalt chloride, or 100 nm cobinamide. Plates were incubated at 37 °C for 24 h. Formation of red colonies indicated the utilization of 1,2-propanediol by the reduction in pH due to the formation of propionate, and a more rapid appearance of red color with the addition of cobinamide indicated cobalamin dependence of propanediol utilization. Isolation of Metabolosomes—Metabolosomes were isolated by the organelle purification procedure as described previously (7Havemann G.D. Bobik T.A. J. Bacteriol. 2003; 185: 5086-5095Crossref PubMed Scopus (127) Google Scholar) and outlined further in the supplemental material. Electron Microscopy—Late logarithmic phase cells of E. coli pLA2917 and pAR3114 were prefixed in 2.5% (v/v) glutardialdehyde, 10 mm Hepes, pH 7.0, for 72 h. After immobilization in 2% (w/v) water-agar they were postfixed in 1% (w/v) osmium tetroxide in 75 mm cacodylate, pH 7.2, for 60 min at 4 °C followed by dehydration in an ethanol series. The 70% dehydration step, supplemented with 1% (w/v) uranylacetate, was done overnight at ambient temperature following the method of Bobik et al. (6Bobik T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar). Ultrathin sections (90 nm) were post-stained with 4% (w/v) aqueous uranylacetate and analyzed at zero-loss bright field mode in an energy-filtered transmission electron microscope (Zeiss CEM 902, Oberkochen, Germany). Isolated polyhedral bodies were fixed in 1% (v/v) glutardialdehyde, and after adsorption to Formvar-carbon-coated grids they were negatively stained with 2% (w/v) uranylacetate, pH 4.5. Samples were analyzed by energy-filtered transmission electron microscope, and images were recorded, in general, with a charge-coupled device camera (CCD; Proscan Electronic Systems, Scheuring, Germany). Fast Fourier transformations (FFT) of individual particles was done with CRISP software (Calidris, Sollentuna, Sweden). SuperSTEM microscopy was carried out on a VG HB501 dedicated STEM fitted with a Nion second generation spherical aberration corrector, high angle annular dark field detector (angular range 70–210 mrad), and a Gatan Enfina electron energy loss spectrometer. Diol Dehydratase Assay—The activity of diol dehydratase was measured by the 3-methyl-2-benzothiazolinone hydrazone method as described previously (23Toraya T. Honda S. Fukui S. J. Bacteriol. 1979; 139: 39-47Crossref PubMed Google Scholar). One unit of diol dehydratase activity is defined as the amount of enzyme that catalyzes the formation of 1 μmol of propionaldehyde/min/mg of protein. EPR Analysis and Redox Potentiometry—Continuous wave EPR spectra were recorded on a Bruker Elexis E500/580 spectrometer operating at X-band (9.7 MHz) equipped with an Oxford helium flow cryostat as described previously. Redox titrations were performed in a Belle Technology glove-box under a nitrogen atmosphere, essentially as described previously (24Munro A.W. Noble M.A. Robledo L. Daff S.N. Chapman S.K. Biochemistry. 2001; 40: 1956-1963Crossref PubMed Scopus (146) Google Scholar, 25Ost T.W. Miles C.S. Munro A.W. Murdoch J. Reid G.A. Chapman S.K. Biochemistry. 2001; 40: 13421-13429Crossref PubMed Scopus (105) Google Scholar). Utilization of 1,2-Propanediol—Transformation of E. coli JM109 cells with a cosmid library made from a partial digest of the C. freundii genome resulted in the identification of a single red colony when the library was plated on propanediol-MacConkey medium (PM medium). The appearance of this red colony was likely due to the ability of the recombinant strain to metabolize 1,2-propanediol, a phenotype absent in wild-type E. coli. The cosmid, termed pAR3114, was found to house an ∼30 kb insert. The insert was sequenced, which revealed that it encoded 30 contiguous genes involved in cobalamin biosynthesis and propanediol utilization and five other genes localized at one end of the insert. These genes and the putative functions of the encoded protein products are described in Table 1. The sequence data have been deposited in the EMBL Nucleotide Sequence Database under accession number AM498294.TABLE 1Genes present on pAR3114, a cosmid containing a 30-kb insert of C. freundii genomic DNAGeneStartEndFunctionLengthDirection of transcriptionPredicted massbpDacbiB8351Cobalamin biosynthesis protein834-17,817cbiA2,211832Cobyrinic acid a,c-diamide synthase1379-49,669pocR3,7182,807Transcriptional regulator911-49,636pduF4,7293,920Propanediol diffusion facilitator809-28,125pduA5,2555,532Shell protein277+9,376pduB5,5366,348Shell protein812+28,021pduC6,3678,031Diol dehydratase large subunit1664+60,280pduD8,0428,716Diol dehydratase medium subunit674+24,280pduE8,7319,249Diol dehydratase small subunit518+19,233pduG9,26511,097Diol dehydratase reactivation protein1832+64,163pduH11,08711,437Diol dehydratase reactivation protein350+12,632pduJ11,45711,732Shell protein275+9,053pduK11,82312,227Shell protein404+13,921pduL12,22712,859Phosphotransacylase632+23,048pduM12,85613,347Unknown491+18,175pduN13,35113,626Shell protein275+9,205pduO13,63514,642Cobalamin adenosyltransferase1007+45,886pduP14,63916,024CoA-dependent propionaldehyde dehydrogenase1385+48,671pduQ16,03517,147Propanol dehydrogenase1112+39,702pduS17,14418,499Cobalamin reductase1355+48,568pduT18,50219,056Shell protein554+19,038pduU19,05619,406Shell protein350+12,483pduV19,41119,863Unknown452+16,331pduW19,84821,062Propionate kinase1214+43,634pduX21,09222,021Unknown929+33,908yeeA23,69422,636Putative inner membrane protein1058-39,618DacD25,54424,372DD-carboxypeptidase1172-43,085phsC26,45025,683Thiosulfate reductase cytochrome B subunit767-28,545phsB27,03526,456Thiosulphate reductase protein579-21,622phsA28,61827,043Thiosulphate reductase precursor1575-53,778 Open table in a new tab Gene Order and Similarity to S. enterica—The C. freundii pdu genes constitute a probable regulon, comprising a 21-gene operon and a regulatory gene. The gene order was found to be identical to that previously reported in S. enterica (6Bobik T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar), forming a divergent operon with the cbi operon for cobinamide synthesis from a central regulatory gene, pocR (Fig. 1a). Subcloning of the pdu Operon—E. coli pAR3114 formed red colonies on the PM plate in the presence of cobinamide, whereas it remained yellow in its absence indicating the requirement of B12 for propanediol metabolism (Fig. 1b). The E. coli pLA2917 vector control strain formed yellow colonies on PM agar with cobinamide, showing its inability to metabolize 1,2-propanediol. The insert within pAR3114 was trimmed in size by deleting the cbiB, cbiA, pocR, and pduF genes upstream of the main pdu operon, and a further five genes downstream of the pdu operon that are transcribed in the opposite direction and are not thought to be associated with the pdu operon (see Table 1). This shortened insert, containing just the 21 genes of the pdu operon, was cloned into pET14 generating plasmid pED460. When transformed into JM109, bacterial colonies turned red when grown on PM indicator plates, demonstrating that the transformed bacteria had gained the ability to metabolize propanediol. A further deletion was made whereby pduA and pduB were removed from the insert in pED460 to give pED461 (supplemental Table S1). The resulting plasmid, when transformed into E. coli JM109, had a greatly reduced ability to metabolize propanediol on PM medium in that the colonies turned only slightly pink. Reconstitution of propanediol metabolism was achieved by adding pduA-B back in trans on a separate, compatible plasmid (pLysS). However, the individual addition of either pduA or pduB did not restore propanediol metabolism, demonstrating that both proteins are required for this activity. Electron Microscopy—Cobalamin-dependent propanediol utilization is associated with the formation of intracellular inclusions called carboxysome-like bodies, enterosomes, or metabolosomes (6Bobik T.A. Havemann G.D. Busch R.J. Williams D.S. Aldrich H.C. J. Bacteriol. 1999; 181: 5967-5975Crossref PubMed Google Scholar). We therefore examined our transformed cells for evidence of metabolosomes. Ultrathin sections of E. coli pAR3114 grown on minimal medium in the presence of 1,2-propanediol showed intracellular metabolosomes (Fig. 2b). E. coli pLA2917 (empty cosmid) control strain grown on minimal medium in the presence of 1,2-propanediol did not show metabolosomes inside the cells (Fig. 2a). When grown on rich medium (LB), E. coli pAR3114 contained metabolosomes in the presence or absence of added propanediol. In cells grown on rich medium, the metabolosomes were closely packed and formed up to 90% of the observed section surface. Again, control strains containing the empty cosmid did not reveal any such structures when grown under similar conditions. In ultrathin cell sections, the metabolosomes measured on average 101 nm across their widest diameter (n = 63; S.D. = 25 nm). There were two maxima in the frequency distribution of measured diameters, compatible with two different size bodies or different sections of a non-spherical polyhedral structure with shorter (77–92 nm) and longer (108–123 nm) axial dimensions (Fig. 2d). These dimensions are similar to those reported for wild-type 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 variable sizes with two distinct maximal diameters (18Schmid M.F. Paredes A.M. Khant H.A. Soyer F. Aldrich H.C. Chiu W. Shively J.M. J. Mol. Biol. 2006; 364: 526-535Crossref PubMed Scopus (110) Google Scholar). When looked at in detail (Fig. 2c, white arrows), apparent sections of polyhedral bodies showed a weak linearly ordered substructure, indicating a certain degree of interior package order. Additionally, polar deposits of electron-dense amorphous granules could be observed in many recombinant cells (Fig. 2b, asterisks). When the E. coli strain containing the pdu operon, but missing pduA-B, was analyzed by electron microscopy, no metabolosomes were observed (data not shown). Metabolosomes were observed again when this strain was complemented with a compatible plasmid containing pduA-B (data not shown). However, no organelles or other structures were seen in strains in which only pduA or pduB was added back in trans (data not shown). This result indicates that both PduA and PduB are required for organelle biogenesis. Previous research on the S. enterica pdu operon had shown that PduA is essential for organelle formation (26Havemann G.D. Sampson E.M. Bobik T.A. J. Bacteriol. 2002; 184: 1253-1261Crossref PubMed Scopus (112) Google Scholar). Diol Dehydratase Activity—The activity of diol dehydratase was measured from the protein extracted from E. coli pAR3114 grown to stationary phase in the presence and absence of 1,2-propanediol. Diol dehydratase activity was detected at 2.23 units/mg whole-cell protein for E. coli pAR3114 grown in the presence of 1,2-propanediol. Activity was reduced to 0.72 unit/mg when E. coli pAR3114 was grown in the absence of 1,2-propanediol. Recombinant metabolosomes were extracted from cells grown in minimal medium with propanediol as per published methods for the extraction of wild-type metabolosomes (7Havemann G.D. Bobik T.A. J. Bacteriol. 2003; 185: 5086-5095Crossref PubMed Scopus (127) Google Scholar). Purified metabolosomes derived from E. coli pAR3114 grown in the presence of 1,2-propanediol showed diol dehydratase activity of 32.03 units/mg protein, ∼14 times higher than the enzyme activity found in the crude protein extract. Extracted Polyhedral Metabolosomes—Metabolosomes were purified from E. coli pAR3114 as described under "Experimental Procedures" and in the supplemental material and were viewed both by an energy-filtered transmission electron microscope in the elastic bright field mode and a novel aberration-corrected non-contrast electron microscopy technique using a SuperSTEM instrument. No such structures were obtained from E. coli control strains, indicating that the metabolosomes were generated as a result of the presence of the pdu operon. The interior of negatively stained polyhedral bodies of E. coli pAR3114, fixed with glutardialdehyde (Fig. 3a), showed a finely branching substructure. These particles had a mean diameter of 92 nm (n = 143; S.D. = 32 nm) with a frequency distribution showing 1 maxima (presumed short axis) from 62 to 77 nm and a presumed long axis from 108 to 123 nm (Fig. 2e). Higher magnifications of those particles showed regularly arranged substructures with a layer spacing of about 7 nm (Fig. 3b, white arrows). Intrinsic order on or below this scale within isolated polyhedral bodies was detected by FFT (Fig. 3c, FFTs 2 and 3) relative to the amorphous foil substratum (Fig. 3c, FFT 1) Distinct frequency intensities within a 128 × 128 matrix were obvious at 0.30 and 0.41 per nm, corresponding to a particulate resolution of 3.3 and 2.4 nm (Fig. 3c, FFT 2). That is, the electron micrographs shown resolve structures as small as 2.4 nm, confirming their ability to detect 7-nm layers. Further evidence for regular metabolosome substructure was obtained using SuperSTEM microscopy of the recombinant metabolosomes both in cell sections and protein extracts. A number of particles displayed apparent multicomponent substructure, with smaller particles containing electron density typical of proteins (Fig. 4, a and b). This suggests that the interior of the metabolosome is not an amorphous mixture of component molecules but constitutes a regular assembly of structural proteins. Such a regular assembly has recently been reported for carboxysomes from Synechocystis, where the RuBisCOs are organized into three or four concentric layers (19Iancu C.V. Ding H.J. Morris D.M. Dias D.P. Gonzales A.D. Martino A. Jensen G.J. J. Mol. Biol. 2007; 372: 764-773Crossref PubMed Scopus (116) Google Scholar). Currently crystal structures of carboxysome and metabolosome shell proteins show them forming flat hexagonal sheets, although it has not been determined how this planar structure can bend or fold up to form a closed shell (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, 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). Our SuperSTEM images showing discrete electron-dense foci spaced around the recombinant metabolosomes suggest the presence of non-planar protein complexes acting as structural linkage points joining sheets of shell protein to form the faceted surface of the metabolosome (Fig. 4, c and d) in the same way that vertex proteins link planar proteins to form the viral capsid structure (27Abrescia N.G. Cockburn J.J. Grimes J.M. Sutton G.C. Diprose J.M. Butcher S.J. Fuller S.D. San Martin C. Burnett R.M. Stuart D.I. Bamford D.H. Bamford J.K. Nature. 2004; 432: 68-74Crossref PubMed Scopus (224) Google Scholar, 28Fokine A. Leiman P.G. Shneider M.M. Ahvazi B. Boeshans K.M. Steven A.C. Black L.W. Mesyanzhinov V.V. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7163-7168Crossref PubMed Scopus (172) Google Scholar). As in viral capsids, these complexes are presumably pentameric, connecting planes of hexameric shell proteins (27Abrescia N.G. Cockburn J.J. Grimes J.M. Sutton G.C. Diprose J.M. Butcher S.J. Fuller S.D. San Martin C. Burnett R.M. Stuart D.I. Bamford D.H. Bamford J.K. Nature. 2004; 432: 68-74Crossref PubMed Scopus (224) Google Scholar, 28Fokine A. Leiman P.G. Shneider M.M. Ahvazi B. Boeshans K.M. Steven A.C. Black L.W. Mesyanzhinov V.V. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7163-7168Crossref PubMed Scopus (172) Google Scholar). The likely candidate for this metabolosome component is one (or more) of the three predicted shell proteins, just as minor variants of viral capsid proteins normally forming hexameric sheets form pentameric vertex proteins (28Fokine A. Leiman P.G. Shneider M.M. Ahvazi B. Boeshans K.M. Steven A.C. Black L.W. Mesyanzhinov V.V. Rossmann M.G. Proc
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