Characterization of intact subcellular bodies in whole bacteria by cryo‐electron tomography and spectroscopic imaging

We illustrate the combined use of cryo‐electron tomography and spectroscopic difference imaging in the study of subcellular structure and subcellular bodies in whole bacteria. We limited our goal and focus to bodies with a distinct elemental composition that was in a sufficiently high concentration to provide the necessary signal‐to‐noise level at the relatively large sample thicknesses of the intact cell. This combination proved very powerful, as demonstrated by the identification of a phosphorus‐rich body in Caulobacter crescentus. We also confirmed the presence of a body rich in carbon, demonstrated that these two types of bodies are readily recognized and distinguished from each other, and provided, for the first time to our knowledge, structural information about them in their intact state. In addition, we also showed the presence of a similar type of phosphorus‐rich body in Deinococcus grandis, a member of a completely unrelated bacteria genus. Cryo‐electron microscopy and tomography allowed the study of the biogenesis and morphology of these bodies at resolutions better than 10 nm, whereas spectroscopic difference imaging provided a direct identification of their chemical composition.

[1]  J. Amalvy,et al.  Elemental mapping by ESI–TEM, during styrene emulsion polymerization , 2001 .

[2]  G. Dellaire,et al.  Organization of chromatin in the interphase mammalian cell. , 2005, Micron.

[3]  J. Frank.  Three-Dimensional Electron Microscopy of Macromolecular Assemblies , 2006 .

[4]  A. Kornberg,et al.  Polyphosphate kinase from Escherichia coli. Purification and demonstration of a phosphoenzyme intermediate. , 1990, The Journal of biological chemistry.

[5]  M. Kikuchi,et al.  Radiation response mechanisms of the extremely radioresistant bacterium Deinococcus radiodurans. , 2004, Uchū Seibutsu Kagaku.

[6]  R. Egerton,et al.  Electron Energy-Loss Spectroscopy in the Electron Microscope , 1995, Springer US.

[7]  B. Little,et al.  Electron energy loss spectroscopy techniques for the study of microbial chromium(VI) reduction. , 2002, Journal of microbiological methods.

[8]  A. Kornberg,et al.  Inorganic Polyphosphate Is Required for Motility of Bacterial Pathogens , 2000, Journal of bacteriology.

[9]  J. T. Staley,et al.  In Situ Reproductive Rate of FreshwaterCaulobacter spp , 2000, Applied and Environmental Microbiology.

[10]  Brad J Marsh,et al.  Lessons from tomographic studies of the mammalian Golgi. , 2005, Biochimica et biophysica acta.

[11]  T. Barkay,et al.  Monitoring of microbial metal transformations in the environment. , 2005, Current opinion in biotechnology.

[12]  Lucy Shapiro,et al.  A Bacterial Cell-Cycle Regulatory Network Operating in Time and Space , 2003, Science.

[13]  H. Santos,et al.  Electron-dense granules in Desulfovibrio gigas do not consist of inorganic triphosphate but of a glucose pentakis(diphosphate). , 1996, European journal of biochemistry.

[14]  A. Kornberg.  Inorganic polyphosphate: toward making a forgotten polymer unforgettable , 1995, Journal of bacteriology.

[15]  Arthur Kornberg,et al.  Inorganic polyphosphate in the origin and survival of species. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Kenneth H Downing,et al.  Dose tolerance at helium and nitrogen temperatures for whole cell electron tomography. , 2005, Journal of structural biology.

[17]  A. Kornberg,et al.  Metaphosphate synthesis by an enzyme from Escherichia coli. , 1956, Biochimica et biophysica acta.

[18]  P. Midgley,et al.  3D electron microscopy in the physical sciences: the development of Z-contrast and EFTEM tomography. , 2003, Ultramicroscopy.

[19]  J. Battista,et al.  A ring-like nucleoid is not necessary for radioresistance in the Deinococcaceae , 2005, BMC Microbiology.

[20]  A. Heinrichs.  Transcription: Signal to reshape , 2003, Nature Reviews Molecular Cell Biology.

[21]  S. Salzberg,et al.  Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. , 1999, Science.

[22]  T. Kornberg.  Another arrow in the Drosophila quiver , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  A. Galembeck,et al.  Scanning electric potential microscopy imaging of polymers: electrical charge distribution in dielectrics , 2001 .

[24]  Maryann E Martone,et al.  Automated most-probable loss tomography of thick selectively stained biological specimens with quantitative measurement of resolution improvement. , 2004, Journal of structural biology.

[25]  W. Baumeister,et al.  Macromolecular Architecture in Eukaryotic Cells Visualized by Cryoelectron Tomography , 2002, Science.

[26]  I. Friedberg,et al.  Structures Containing Polyphosphate in Micrococcus lysodeikticus , 1968, Journal of bacteriology.

[27]  J. N. Parle.  Micro-Organisms in the Intestines of Earthworms , 1963 .

[28]  M Marko,et al.  The Emergence of Electron Tomography as an Important Tool for Investigating Cellular Ultrastructure , 2001, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[29]  Wolfgang Baumeister,et al.  Electron tomography: towards visualizing the molecular organization of the cytoplasm. , 2002, Current opinion in structural biology.

[30]  J. Blum,et al.  Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major. , 1990, Molecular and biochemical parasitology.

[31]  A. Kornberg,et al.  A polyphosphate kinase (PPK2) widely conserved in bacteria , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[32]  K. Gezelius.  Inorganic polyphosphates and enzymes of polyphosphate metabolism in the cellular slime mold Dictyostelium discoideum , 1974, Archives of Microbiology.

[33]  J. Golecki,et al.  Ultrastructural and electron spectroscopic analyses of cyanobacteria and bacteria , 1991, Journal of microscopy.

[34]  G. Dellaire,et al.  Correlative light and electron spectroscopic imaging of chromatin in situ. , 2004, Methods in enzymology.

[35]  J. Poindexter,et al.  Combined procedure for assays of poly-β-hyroxybutyric acid and inorganic polyphosphate , 1983 .

[36]  W. Baumeister,et al.  Perspectives of molecular and cellular electron tomography. , 1997, Journal of structural biology.

[37]  A. Earl,et al.  Why is Deinococcus radiodurans so resistant to ionizing radiation? , 1999, Trends in microbiology.

[38]  Y. Amano,et al.  Electron Microprobe X‐Ray Analysis of Polyphosphate Granules in Plesiomonas shigelloides , 1987, Microbiology and immunology.

[39]  Judith Klumperman,et al.  Electron microscopy in cell biology: integrating structure and function. , 2003, Nature reviews. Molecular cell biology.

[40]  W. A. Corpe,et al.  Quantitative analysis of the elemental composition and the mass of bacterial polyphosphate bodies using STEM EDX. , 2001, Microbios.

[41]  Y. Matsumoto,et al.  Microbeams of heavy charged particles. , 2004, Uchu Seibutsu Kagaku.

[42]  Ian T. Paulsen,et al.  Complete genome sequence of Caulobacter crescentus , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[43]  R. Leapman,et al.  Three-dimensional distributions of elements in biological samples by energy-filtered electron tomography. , 2004, Ultramicroscopy.

[44]  W. Stewart.  Nitrogen-fixing cyanobacteria and their associations with eukaryotic plants , 1978 .

[45]  Peter J Lewis.  Bacterial subcellular architecture: recent advances and future prospects , 2004, Molecular microbiology.

[46]  Q. Qi,et al.  Polyhydroxybutyrate biosynthesis in Caulobacter crescentus: molecular characterization of the polyhydroxybutyrate synthase. , 2001, Microbiology.

[47]  Mason R. Mackey,et al.  Cometabolism of Cr(VI) by Shewanella oneidensis MR‐1 produces cell‐associated reduced chromium and inhibits growth , 2003, Biotechnology and bioengineering.