Pyrodictium cannulae enter the periplasmic space but do not enter the cytoplasm, as revealed by cryo-electron tomography
Introduction
Pyrodictium was the first hyperthermophilic prokaryote for which growth at temperatures above 100 °C could be demonstrated (Stetter, 1982, Stetter, 1996). All Pyrodictium strains known today are able to grow at temperatures between 75 and 110 °C at neutral pH under strictly anaerobic conditions by sulfur-hydrogen autotrophy (Dirmeier et al., 1998; Pley et al., 1991; Stetter et al., 1983). The surface of the cells is covered by a two-dimensional (2D) protein array (S-layer) with hexagonal symmetry. The dome-shaped hexamers are anchored to the cytoplasmic membrane by stalks, or filiform protrusions, thereby spanning the periplasmic space with a constant width of approximately 35 nm (Baumeister and Lembcke, 1992; Dürr et al., 1991).
Pyrodictium cells synthesise a peculiar extracellular matrix in which the cells are entrapped (König et al., 1988; Rieger et al., 1995). It consists of bundles of hollow tubules, the cannulae, which have an outer diameter of 25 nm and are made up of (at least) three homologous glycoproteins. A view of the dynamics of this network and the cells in vivo was obtained by observing their growth at 90 °C under anoxic conditions, using a high-intensity dark-field light microscope: cell division and growth of the cannulae were seen to be directly linked (Horn et al., 1999). When the daughter cells separate after cell division, they remain connected by cannulae; by multiple repetition of this process, a colony of cells develops which is interconnected by a dense network, with each cell exhibiting multiple connections with its neighbours. In the same study, single cannulae with one free end could be observed; they were not connected to a second cell, but grew freely into the culture medium.
Two previous studies focused on the ultrastructure of Pyrodictium cells and the cannulae network (Rieger et al., 1995, Rieger et al., 1997). In ultrathin sections, cannulae were visible in the cell-free medium only, but not inside the cell, neither in the periplasm nor in the cytoplasm. Scanning electron microscopy (SEM) images revealed the network of cannulae and the cells in toto. Apparently, the cannulae insert into the cells at various points scattered all over the cell surface, rather than at one or few preferential sites. Transmission electron microscopy (TEM) studies of freeze-etched cells indicated that the cannulae may enter the periplasmic space. However, both techniques give insight only into the surface of objects; they do not allow analysis of how far the cannulae reach into the cell interior.
Electron tomography (ET) is the only EM-based technique which can provide three-dimensional images of large and pleiomorphic biological structures (Baumeister and Steven, 2000; Frank, 1992; Koster et al., 1992, Koster et al., 1997). In contrast, a single TEM micrograph only provides a two-dimensional projection image of a three-dimensional (3D) volume: features of the object that lie in the direction of the electron beam do not become separated in the final image. With the advent of automated data acquisition it became possible to apply ET to radiation-sensitive specimens such as biological materials embedded in amorphous ice. Thus it is now possible to combine the power of 3D imaging with a close-to-life preservation of the specimen (Dierksen et al., 1995; Grimm et al., 1997; Nicastro et al., 2000), even of unfixed, fully hydrated prokaryotic cells, such as Sulfolobus and Pyrobaculum (Grimm et al., 1998).
Here, we have employed cryo-electron tomography for visualizing the interaction site of a Pyrodictium abyssi cell with its cannulae. In particular, we aimed at determining whether the cannulae enter the cytoplasm, thereby crossing the S-layer and the cytoplasmic membrane, or whether they only enter the periplasm. To minimise distortion due to the limited tilt range we have used dual-axis tilting by using a newly designed cryo-holder (Nickell et al., unpublished). The reconstructed volume provided the first visualisation of (i) the cannulae inside a Pyrodictium cell, (ii) discrete particles in the periplasm, and (iii) “empty” and “filled” cannulae. The fact that the cannulae are located exclusively in the extracellular and periplasmic space has implications on considerations about their putative function(s).
Section snippets
Materials and methods
Cultivation and preparation of cells. Cells of P. abyssi strain TAG11 (Rieger et al., 1995) were cultivated in modified SME medium (Stetter et al., 1983), containing , and a gas phase consisting of N2:CO2 (80:20; 300 kPa) as described (Pley et al., 1991). Cells were concentrated by low-speed centrifugation (5000g) in a table-top centrifuge (Beckman microfuge 11) and resuspended in 100 μl culture medium. A droplet of the suspension was applied to a copper grid covered with a holey carbon
Data recording
In this study, we analysed the structural details visible in the reconstructed volume of a Pyrodictium cell. It was embedded in vitreous ice on a holey carbon film (Fig. 1). Because in single axis tilt series, the “missing wedge” results in blurring or even obscuring important structural details, we have, for the first time, utilised a cryo-holder (Nickell S., Hegerl R., Armbruster B., Baumeister W., unpublished) which allowed two-axis tilting of the same specimen area (Fig. 1). Merging the two
Supplementary Files
Acknowledgements
We thank Prof. Dr. K. O. Stetter for continuous support, Peter Hummel for growing Pyrodictium cells, and Dr. Dieter Typke for stimulating discussions. For the construction of the rotation-tilt cryo-holder we thank Rudolf Gatz from the Department of Structural Biology, MPI für Biochemie, Martinsried, and Dr. Barbara Armbruster and Ron Zolkowski (both Gatan Inc., Pleasanton, CA) for support during the designing process. We also thank Dr. Brian Hedlund for carefully reading the manuscript. This
References (39)
- et al.
Macromolecular electron microscopy in the era of structural genomics
Trends Biochem. Sci.
(2000) - et al.
Toward automatic electron tomography
Ultramicroscopy
(1992) - et al.
Toward automatic electron tomography. II. Implementation of autofocus and low-dose procedures
Ultramicroscopy
(1993) - et al.
Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography
Biophys. J.
(1995) - et al.
Three-dimensional reconstruction of the surface protein of Pyrodictium brockii: comparing two image processing strategies
J. Struct. Biol.
(1991) - et al.
Determination of the inelastic mean free path in ice by examination of tilted vesicles and automated most probable loss imaging
Ultramicroscopy
(1996) - et al.
Energy-filtered electron tomography of ice-embedded actin and vesicles
Biophys. J.
(1997) - et al.
Electron tomography of ice-embedded prokaryotic cells
Biophys. J.
(1998) The EM program package: A platform for image processing in biological electron microscopy
J. Struct. Biol.
(1996)- et al.
The tripartite ATP-dependent periplasmic (TRAP) transporters of bacteria and archaea
FEMS Microbiol. Rev.
(2001)
The fine structure of the fibres of Pyrodictium occultum
FEMS Microbiol. Lett.
Automated microscopy for electron tomography
Ultramicroscopy
Perspectives of molecular and cellular electron tomography
J. Struct. Biol.
An imaging filter for biological applications
Ultramicroscopy
Dual-axis tomography: an approach with alignment methods that preserve resolution
J. Struct. Biol.
A hyperthermostable protease of the subtilisin family bound to the surface layer of the archaeon Staphylothermus marinus
Curr. Biol.
Cryo-electron tomography of Neurospora mitochondria
J. Struct. Biol.
Double-tilt electron tomography
Ultramicroscopy
Pyrodictium abyssi sp. nov. represents a novel heterotrophic marine archaeal hyperthermophile growing at 110 °C
Syst. Appl. Microbiol.
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2013, Journal of Structural BiologyCitation Excerpt :As a consequence, the resolution in the plane of the specimen becomes more isotropic, since information lacking perpendicular to the tilt axis in the first series can be retrieved from the second one. Although dual-axis electron tomography has proved to be very efficient in minimizing imaging artifacts induced by the missing wedge on fixed specimens embedded in resin, it has only been sparsely used with samples vitrified in their close-to native state (Benjamin et al., 2005; Dudkina et al., 2010; Iancu et al., 2005; Komeili et al., 2006; Murphy and Jensen, 2005; Nickell et al., 2003). Amongst possible explanations, one could be technical limitations due to the available equipment or to the time required to acquire dual-axis tilt series of vitrified specimens.