FEMS Microbiology Reviews 20 (1997) 13^23
II. Fine structure of S-layers 1
í marda 3;c , David S
í majs c ,
Reinhard Rachel 2;3;a , Dietmar Pum 2;b , Jan S
è nek d , Gertraud Rieger a , Karl O. Stetter a
Jir|è Komrska d , Vladislav Krzyza
b
a
Lehrstuhl fuër Mikrobiologie und Archaeenzentrum, Universitaët Regensburg, D-93040 Regensburg, Germany
Zentrum fuër Ultrastrukturforschung und Ludwig Boltzmann-Institute fuër Molekulare Nanotechnologie, Universitaët fuër Bodenkultur,
A-1180 Vienna, Austria
c
Department of Biology, Faculty of Medicine, Masaryk University, CZ-66243 Brno, Czech Republic
d
Institute of Physical Engineering, Faculty of Mechanical Engineering, Technical University, CZ-61669 Brno, Czech Republic
Received 10 March 1997 ; accepted 24 March 1997
Abstract
S-layers are now considered a common cell wall structure in Bacteria and Archaea as well as in some algae. Morphological
and chemical studies have revealed that S-layers consist of crystalline arrays of protein or glycoprotein subunits forming
oblique, square or hexagonal lattices on the cell surface. Electron microscopy and computer image enhancement techniques
have been applied to obtain structural information down to the nanometer range. This chapter deals with the wide distribution
of S-layers among cyanobacteria, and their morphological and chemical characterization, and the potential of high resolution
electron microscopic studies applied to the cell envelope of
Pyrodictium.
The occurrence of S-layers in cyanobacteria was
investigated by cryomethods and ultrathin sectioning. These investigations indicate that the ultrastructure of S-layers may be
exploited as an auxiliary taxonomic criterion in the classification of cyanobacteria.
Pyrodictium is the first organism which has
shown an optimum growth temperature above 100³C. The highly irregularly shaped, flagellated cells are interconnected by
extracellular tubules. The three-dimensional structure of this network was visualized with high resolution scanning electron
microscopy while the fine structure of the cell wall architecture was studied in detail with various electron microscopic
techniques. Both contributions demonstrate that the investigation of the fine structure of S-layers is fundamental for
establishing structure-function relationships for these two-dimensional crystalline arrays.
Keywords:
Cyanobacteria ; S-layer ; Hyperthermophilic Archaea ; Cell wall ;
Pyrodictium ;
Laser di¡ractometry ; Cryo-preparation
1
This review is part of a series of reviews dealing with different aspects of bacterial S-layers ; all these reviews appeared in Volume
20/1-2 (June 1997) of
, thematic issue devoted to bacterial S-layers.
FEMS Microbiology Reviews
2
Guest Editor.
3
Corresponding authors. Dr. Reinhard Rachel : Tel. : +49 (941) 943-4534 ; Fax : +49 (941) 943-2403 ; E-mail : [email protected]. Prof. Dr. Jan Smarda : Tel. : +42 (5) 421 26 259 ; Fax : +42 (5) 421 26 200 ; E-mail : [email protected].
0168-6445 / 97 / $32.00 ß 1997 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
PII S 0 1 6 8 - 6 4 4 5 ( 9 7 ) 0 0 0 4 0 - 5
14
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. Twenty-¢ve years of experience on S-layers of cyanobacteria: occurrence and patterns . . . . . . .
2.1. Occurrence of S-layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Morphological and chemical characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.1. S-layer morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.2. Chemical composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.3. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3. Ultrastructure of hyperthermophilic Archaea: cryo-preparation methods applied to Pyrodictium
3.1. Cryo-preparation methods for prokaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. The ultrastructure of Pyrodictium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
S-layers (surface-layers) composed of protein subunits are common cell wall structures in Bacteria,
Archaea, and even some algae. They form the outer
layer of these cell walls facing the surrounding medium. This review deals with the ¢ne structure of
S-layers, mainly of cyanobacteria and of the hyperthermophilic archaebacterial genus Pyrodictium, as
revealed by electron microscopic and other techniques. These techniques include freeze-etching, ultrathin sectioning, cryo-electron microscopy, optical
(laser) di¡raction crystallography, two-dimensional
reconstruction of digitized di¡raction pattern following Fourier transformation, and other advanced
techniques of light and electron microscopy, in combination with genetic studies.
These studies reveal the crystalline monomolecular assembly of the protein or glycoprotein subunits as marvellous porous lattices completely covering the cell surface. Understanding the ¢ne
structure of these layers is a prerequisite for further
understanding the biochemistry and function of
these cell structures as well as for potential applications.
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21
2. Twenty-¢ve years of experience on S-layers of
cyanobacteria: occurrence and patterns
Jan Símarda3 , David Símajs, Jir|è Komrska,
Vladislav Krzyzaènek
The ¢rst observation of a regular structure on the
cell surface of a cyanobacterial cell was reported in
Gloeocapsa alpicola in 1972 [1,2]. Schiewer and
Jonas, studying the ultrastructure of cells of the Synechocystis aquatilis strain Gromov/Len. 428 in
transverse sections, observed ``a distinct texture
formed by electron-dense and electron-transparent
radial rows between two electron-dense border
layers'' in the outermost cell wall layer. During the
following 20 years, several authors observed many
more S-layers in dozens of di¡erent cyanobacterial
strains, using mainly freeze-etching and freeze-fracturing procedures. The morphology of the S-layers
was investigated in detail with electron microscopy,
laser di¡raction analysis, and Fourier transformation
followed by two-dimensional reconstructions of the
digitized patterns [3^14].
An overview on the occurrence and the molecular
architecture of S-layers in cyanobacteria after 25
years of research is given here. Several articles have
been published since [3^14].
2.1. Occurrence of S-layers
Several species and strains of cyanobacteria have
been analyzed for the presence of S-layers so far.
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
Table 1
Distribution of S-layers in cyanobacteria
Taxon
Strain
Subunit lattice
symmetry spacing (nm)a
Order: Chroococcales
Family: Synechococcaceae
Subfamily: Aphanothecoideae
Aphanothece cf. halophytica
ATCC 29534
P
Cyanothece aeruginosa
Koch 1974
p2
Cyanothece cedrorum
Lefevre 1937/19 (Fig. 1)
p4
Cyanothece cf. minervae
755
p4
Subfamily: Synechococcoideae
Synechococcus bigranulatus
Kovrov 1972/8 (Fig. 2)
p4
Synechococcus rubescens
Chang 1979
p6
Synechococcus sp.
P-11-17
P
Synechococcus sp.
GC
p6
Synechococcus sp.
GL 24
p6
Synechococcus sp.
BS 8901
P
Family: Merismopediaceae
Aphanocapsa incerta
151
p6
Aphanocapsa rivularis
Pringsheim 1947/Camb. 1404-1 p6
Aphanocapsa sp.
6308
P
Merismopedia sp.
Hindaèk 1985/2
P
Synechocystis aquatilis
Gromov/Len. 428
p6
Synechocystis aquatilis
Holubcovaè 1959/1
p6
Synechocystis aquatilis
Fitzgerald 1051 (Fig. 3)
p6
Synechocystis aquatilis
Gromov 1973/14
p6
Synechocystis aquatilis
Kovaèeéik 1990/8
p6
Synechocystis aquatilis
Markle 1430/3 (Fig. 4)
p6
Synechocystis fuscopigmentosa
George 1954/Camb. 1412-4
p6
Synechocystis salina
Vaara 1978/CB-3
p6
Synechocystis cf. salina
n.g.
p6
Synechocystis sp.
CB 3
p6
Synechocystis sp.
CLII
p6
Synechocystis sp.
PCC 6803
p6
Synechocystis sp.
Hindaèk 1985/3
P
Woronichinia naegeliana
n.s. (Karlovy Vary 1994) (Fig. 5) p6
Family: Microcystaceae
Gloeocapsa alpicola
UTEX B 589
P
Gloeocapsa alpicola
Wildman, Bowen 1974
P
Microcystis aeruginosa
n.s. (Finjasjoën 1992) (Fig. 6)
p6
Microcystis aeruginosa
E-3
p6
Microcystis cf. aeruginosa
n.s. (Brno 1993)
p6
Microcystis cf. aeruginosa
n.s. (B|ètov 1993)
p6
Microcystis botrys
NIVA
p6
Microcystis ¢rma
398
p6
Microcystis ¢rma
Schiewer
p6
Microcystis ichthyoblabe
Pas-M£a-1
p6
Microcystis cf. marginata
n.s. (Upper Galilee 1974)
p6
Microcystis wesenbergii
H-M-1
p6
Microcystis wesenbergii
Pas-M-2
p6
Microcystis wesenbergii
Kro-M-5
p6
Microcystis wesenbergii
D-Mw-10
p6
Microcystis cf. wesenbergii
n.s. (B|ètov 1994) (Fig. 7)
p6
Microcystis sp.
691
P
15
Reference
^
8.6
6.9
^
[15]
[10]
[10]
[16]
9.7
^
^
^
22.0
^
[10]
this review
[17]
[3]
[18]
[19]
17.5
12.0
^
^
14.8
15.4
16.3
17.0
17.4
17.6
^
16.4
^
15.2
15.2
^
^
18.6
[5,8]
[6,8]
[6]
[6]
[2,6,8]
[6,20]
[10]
[10]
[10]
[10]
[5]
[6,8]
[5]
[21]
[3,22]
[3]
[6]
this review
^
^
11.5
^
10.7
^
15.4
^
15.6
13.6
14.2
14.3
18.6
14.8
^
14.1
^
[1]
[5]
[10]
this review
[10]
this review
this review
[3]
[8]
this review
[23]
this review
this review
this review
this review
[10]
[6]
16
Table 1 (continued).
Taxon
R. Rachel et al./FEMS Microbiology Reviews 20 (1997) 13^23
Strain
Subunit lattice
symmetry spacing (nm)
Reference
a
Family: Xenococcaceae
Chroococcidiopsis sp.
Buë-S 5105-83
p2
2.5
[24]
Chroococcidiopsis sp.
Buë-26391cPH-80
p2
2.5
[24]
Chroococcidiopsis sp.
n.g.
p2
^
[25]
Order: Oscillatoriales
Family: Pseudanabaenaceae
Subfamily: Leptolyngbyoideae
Leptolyngbya hypolimnetica
n.s. (Athens 1994) (Fig. 8)
p2
9.4
[10]
Family: Phormidiaceae
Subfamily: Phormidioideae
Phormidium cf. uncinatum
n.s. (Tuëbingen)
p4
11.2
L. Ho¡mann (1991) pers. commun.
Phormidium cf. uncinatum
n.s. (Baikal)
p4
11.2
L. Ho¡mann (1991) pers. commun.
Family: Oscillatoriaceae
Subfamily: Oscillatorioideae
Lyngbya cf. aeruginosa
B 47.79
p4
14.0
L. Ho¡mann (1991) pers. commun.
Oscillatoria princeps
n.s. (Nyphenburg 1991)
p4
14.0
L. Ho¡mann (1991) pers. commun.
Data given are average values of several measurements.
cf.: confer; the taxonomic classi¢cation is obsolete or probably incorrect.
P: periodic structure, not characterized in greater detail.
n.g.: not given; n.s.: natural sample, classi¢ed by light microscopy (location of sampling stated in parentheses).
a
Most of them possessed S-layers. The absence of
S-layers in some species can be explained either by
the true absence of functional genes or by the use of
inappropriate cultivation or preparation methods.
Generally, the capability to form an S-layer appears
to be rather genus-speci¢c, whereas an S-layer of a
given lattice symmetry can be considered a common
hereditary marker in a distinct species or strain.
Therefore, it can be used as an auxiliary taxonomic
criterion for the classi¢cation of cyanobacteria. The
results of the previous investigations are summarized
in Table 1.
In summary, S-layers were observed in 56 strains
or isolates of at least 25 species (14 genera, seven
families) of cyanobacteria. Fifty-one strains (21 species, 10 genera) are unicellular organisms of the order Chroococcales, ¢ve strains (four species, four
genera) belong to ¢brillar, multicellular organisms
of the order Oscillatoriales. Though the most recent
classi¢cation system of cyanobacteria is used in this
review, it is subject to further improvements and
continuous development. The taxonomic a¤liation
of some species is still uncertain. For example,
Schiewer's strain Microcystis ¢rma appears, according to several contemporary criteria, much more
closely related to Synechocystis than to Microcystis.
The center-to-center spacing of the hexagonal S-layer
concurs much better with the coarser lattice type of
Synechocystis S-layer. In this context, further reclassi¢cations are to be expected.
On the cells of Synechocystis aquatilis strain
Holubcovaè 1959/1, two superimposed, hexagonal
S-layers were observed. The inner layer showed a
¢ne lattice (15.4 nm spacing), whereas the outer layer
appeared coarser.
Based on the present knowledge, S-layers appear
to be most frequent within the genera Microcystis
and Synechocystis and less frequent in ¢lamentous
C
Fig. 1. Ultrastructure of S-layers of di¡erent cyanobacteria. Bar=100 nm. The insets show the 2-D reconstruction of the S-layer at a
magni¢cation 3 times higher than in the micrographs. A: Cyanothece cedrorum Lefevre 1937/19. B: Synechococcus bigranulatus Kovrov
1972/8. C: Synechocystis aquatilis Fitzgerald 1051. D: Synechocystis aquatilis Markle 1430/3. E: Woronichinia naegeliana Karlovy Vary
1994. F: Microcystis aeruginosa Finjasjoën 1992. G: Microcystis cf. wesenbergii B|ètov 1994. H: Leptolyngbya hypolimnetica Athens 1994.
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
17
18
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
genera. Relatively large cells of planktonic genera,
harboring gas vesicles, usually exhibit S-layers, while
planktonic genera of small cells (without gas vesicles)
appear to have them rarely.
2.2. Morphological and chemical characterization
2.2.1. S-layer morphology
Until now, crystalline S-layers have been determined in 47 cyanobacterial strains. P6 symmetry
was present in 42 unicellular Chroococcales strains,
but p2 and p4 lattices were occasionally found, too.
In contrast, only p2 and p4 lattices were detected in
the ¢ve ¢lamentous Oscillatoriales strains. The data
are summarized in Table 1. From the measurements
it is evident that the lattice constants range from 2.5
to 22 nm, which represents the same dimensions as
for S-layers of Bacteria and Archaea. Five clusters of
lattice constants, from a very ¢ne one to the coarse
lattice, seem to be appropriate for the description:
I: very ¢ne arrays
II: ¢ne arrays
III:medium arrays
IV: coarse arrays
V: very coarse arrays
lattice
lattice
lattice
lattice
lattice
constant
constant
constant
constant
constant
2^3 nm
6^7 nm
8.5^12 nm
13.5^19 nm
21^23 nm
In Fig. 1 di¡erent S-layers from cyanobacteria are
shown. The packing density of the S-layers from individual strains varies within narrow limits. Coarse
S-layers are most frequent. The ¢nest lattice constants occur in p2 lattices, medium lattice constants
are present in p2, p4 and p6 arrays, and large lattice
constants are typical for p4 or p6 symmetry. This is
identical to all other Bacteria. It is typical that all
strains and species of a given genus belong to the
same or to two most closely spacing clusters. Striking exceptions are known only for Synechococcus
bigranulatus (p4 lattice, 9.7 nm spacing) and Synechococcus sp. GL 24 (p6 lattice, 22.0 nm spacing).
Irrespective of all other taxonomic parameters, this
signi¢cant di¡erence clearly indicates that these two
strains do not belong to the same genus and emphasize the hitherto systematic obscurity of the genus
Synechococcus.
Two-dimensional computer image reconstructions
based on Fourier transforms of electron micrographs
were published for Synechococcus sp. GL 24 (p6)
[17,26,27] and for Phormidium cf. uncinatum Baikal
(p4) [25]. 2-D reconstructions of S-layers of Synechococcus bigranulatus (p4), Synechocystis aquatilis (p6),
Woronichinia naegeliana (p6) and Microcystis cf. wesenbergii (p6) are shown, which were reconstructed
from freeze-etched and unidirectionally shadowed
preparations of intact cyanobacterial cells. Interestingly, the morphology of the hexagonal S-layer lattices of Synechocystis and Microcystis as well as
Woronichinia naegeliana is di¡erent.
2.2.2. Chemical composition
Only the S-layer protein of Synechococcus sp.
strain GL 24 has been subjected to chemical analysis,
so far. By SDS-PAGE two proteins, Mr 104 000 and
109 000, were found. When assembled to the intact
lattice, it confers a net neutral charge to the cell surface, and negatively charged sites to the large holes
of the array [18,26].
2.2.3. Conclusions
The summarized data imply that most strains of a
given species possess characteristic S-layer lattices on
their cell surface. The lattice constants seem to be
similar in related strains. Hence, the capability to
form an S-layer of a speci¢c type appears to be a
hereditary marker for a given taxon and can be considered a rather reliable auxiliary taxonomic criterion
in the classi¢cation of cyanobacteria. However, within some genera, considerable di¡erences were noticed
between species. This is an additional argument for
the taxonomic value of S-layers. However, as in Bacteria and Archaea [7], some cyanobacteria do not
possess S-layers. Presumably, such strains can be
considered mutant strains.
The vast majority of the taxa of cyanobacteria
remain to be investigated for the presence and signi¢cance of S-layers on their cell walls. So far, only
one report has been published on the function of
S-layer in cyanobacteria [19]. In the freshwater strain
Synechococcus sp. GL 24, the S-layer acts as a template for mineralization of ¢ne-grain gypsum and
calcite in water with high concentrations of Ca2‡
and SO243 ; its protomers serve as nucleation sites
for the initial mineral deposition.
The basic functions of cyanobacterial S-layers
probably resemble those of other Bacteria. They
may act as: (1) protective coats; (2) molecular sieves
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
and molecule and ion traps; (3) cell adhesion and
surface recognition structures [28]. Additionally,
planktonic cyanobacterial species forming water
blooms may substantially participate in allergic reactions as the causative agent of allergic a¡ections of
human skin (contact eczematous lesions).
3. Ultrastructure of hyperthermophilic Archaea:
cryo-preparation methods applied to Pyrodictium
Reinhard Rachel3 , Gertraud Rieger,
Karl O. Stetter
Pyrodictium was the ¢rst organism which could be
shown to be able to grow optimally at temperatures
above 100³C [29]. Today, Pyrodictium, Methanopyrus [30], and Pyrolobus [31] are the three microorganisms with the highest growth temperature known so
far [32,33]. In the phylogenetic tree, they represent
the deepest and shortest lineages of evolution [32].
Three species of Pyrodictium have been described so
far, with four isolates: P. occultum and P. brockii
[34], P. abyssi [35], and P. abyssi strain TAG 11
[36]. As the organism forms £akes in liquid cultures,
there was an early interest in its ultrastructure. First
investigations were done with conventional electron
microscopic methods, showing that cells of this pro-
Fig. 2. The well preserved three-dimensional structure of the network of
crograph. Bar = 1 Wm.
19
karyote have an unusual, highly irregular shape, possessing extracellular appendages, initially named `¢bers' [29,34,37]. Cells are completely covered by a
surface layer of glycoprotein subunits [34]. The architecture of these two-dimensional crystals was elucidated by electron crystallography [38,39].
Pyrodictium also expresses a thermosome, a chaperonin-like protein complex with ATPase activity,
which is accumulated upon heat shock [40,41]. The
rRNAs and tRNAs are modi¢ed posttranslationally,
probably in response to the high temperature [42^
44]. Pyrodictium possesses two K-like DNA polymerases [45]. Its hydrogen uptake hydrogenase and components for electron transport have been characterized [46,47].
3.1. Cryo-preparation methods for prokaryotes
Most prokaryotic microorganisms have been investigated in the past mainly by three methods: (i)
thin sectioning of cells after room temperature dehydration and conventional embedding; (ii) metal
shadowing or (negative) staining of chemically ¢xed,
air-dried cells; (iii) structural analysis of the cell wall
proteins, in particular of the surface layer protein.
By the application of cryo-preparation techniques,
and, at the same time, by avoiding centrifugation
steps, several new ultrastructural features of Pyrodic-
Pyrodictium cells and extracellular tubules. Scanning
electron mi-
20
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
Fig. 3. Ultrathin section of a
Pyrodictium
cell with an ultra£at area, showing the architecture of the cell envelope. Bar = 0.25
cells were recently observed, using scanning and
transmission electron microscopy [48]. Brie£y, thin
sections were obtained after cryo¢xation and
freeze-substitution [49^51] ; the surface-layer and appendages were visualized after freeze-etching and
also by cryo-electron microscopy [52] ; the threedimensional network of the organism could be visualized by scanning electron microscopy [53].
Freeze-etching can be regarded a standard technique in the investigation of cell surface structures.
Cryo¢xation and freeze-substitution as an alternative
to conventional room temperature dehydration protocols was already introduced many years ago [54^
56] ; it has been used in many studies now, showing
that the ultrastructure of microorganisms is much
better preserved [57^60].
tium
3.2. The ultrastructure of Pyrodictium
The network of Pyrodictium, as it grows in liquid
cultures, could be visualized as a three-dimensional
arrangement of cells, being intertwined with an extensive network of extracellular tubules (Fig. 2) [48].
The shape of the cells was shown to be highly
irregular and lobed, frequently with £at areas. As a
consequence, the surface area of the cells is high,
giving much space in the cell membrane for nutrient
uptake systems, like the one for molecular hydrogen
[56]. Cells possessed two extracellular appendages,
10 nm thin £agella [48,61], and extracellular tubules,
Wm.
which are hollow cylinders with an outer diameter of
approximately 25 nm. The cytoplasm is homogeneous and densely packed, but also occasionally contains regular structures. The cell envelope consists of
a cytoplasmic membrane, a periplasmic space (35 nm
wide), which is not empty, but contains stained material, and a zigzag-shaped surface-layer protein
(Fig. 3).
On the surface of Pyrodictium cells, the S-layer
protein is arranged on a two-dimensional lattice
with p6 symmetry and a center-to-center distance
of 21 nm (Fig. 4) [38,39,62]. The lattice is £exible,
with local distortions as well as ordered regions, depending on the curvature of the cell surface. It is,
however, not as perfectly ordered and rigid as in
Thermoproteus or Pyrobaculum [63,64]. Tubules
were seen to be squeezed in between rows of the
S-layer protein complexes, thereby entering the periplasmic space. They appear to be empty, when crossfractured or thin-sectioned. The tubules are built
from several homologous proteins with a mass of
approximately 20 000 and are extremely heat-resistant, tolerating temperatures up to 140³C for 60 min
in growth medium.
3.3. Conclusions and perspectives
The improved structural preservation of Pyrodicobtained by cryomethods demonstrates that this
organism is highly unusual ; when applied to other
tium
R. Rachel et al. / FEMS Microbiology Reviews 20 (1997) 13^23
21
Fig. 4. Freeze-etched surface of a Pyrodictium cell, with many tubules. Bar = 0.25 Wm.
Archaea, new ultrastructural features are likely to be
detected, changing our view on the structural organizations of these cells.
Pyrodictium as a complex organism can now be
investigated by several approaches, in order to better
understand its functional organization: light microscopy may help to understand how cells divide, and
how they develop the extracellular network of tubules. The attachment of the tubules to the cell can
be clari¢ed by analysis of serial sections or via immunolocalization. The ultrastructure of the tubules
can be analyzed by cryo-electron microscopy and
image processing. The corresponding genes are to
be sequenced, and the regulation of their expression
studied in detail. Finally, if the proteins can be expressed in other organisms, the assembly can be investigated and compared to other structures, like the
microtubules of eukaryotes [65]. Finally, the tubules
will be tested for either intrinsic or associated enzymatic activities.
Acknowledgments
Jan Símarda, David Símajs, Jir|è Komrska and Vladislav Krzyzaènek express their thanks to Professor
Uwe B. Sleytr (Universitaët fuër Bodenkultur, Vienna,
Austria) for his continuous support in freeze-etching
of specimens. They also thank Professor J. Komarak
for his supply of many strains and isolates, guidance in the taxonomy of cyanobacteria and continuous encouragement and criticism. Their work
was supported by grants from the Grant Agency of
the Czech Republic (No. 310/95/1191) and from
the Universities Development Fund of the Ministry of Education of the Czech Republic (No.
0889/1994).
Reinhard Rachel, Gertraud Rieger and Karl O.
Stetter wish to thank R. Hermann (ETH Zuërich)
for scanning electron microscopy, P. Hummel for
expert technical assistance, S. Schneider for her
help in the early phase of this work, R. Schroëder
(MPI Heidelberg), S. Fuller (EMBL Heidelberg)
and S. Graëber for advice and support in cryo-electron microscopy, and C. Brunk for critically reading
the manuscript. Their work was supported by grants
of the DFG (SFB 43 and Ra 751/1-1) and the Fonds
der Chemischen Industrie.
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