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Archebacteria

or
Archaea
?
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Dr.Farokh Rokhbakhsh-Zamin
and Dr.Nadia KAzemiPour

The word archaea comes from the Ancient Greek , meaning


"ancient things", as the first representatives of the domain Archaea were
methanogens and it was assumed that their metabolism reflected Earths primitive
atmosphere and the organisms antiquity.

History:
Until 20th century: Living organisms belongs to 2 kingdoms (Plants & Animals)
In the 1950s & 1960s, previous system failed to accommodate Fungi, Protists &
Bacteria.
By the 1970s, 5 Kingdoms system: 1 prokaryotic (Bacteria) and 4 Eukaryotics
(plants, Animals, Fungi & Protists).
In the late 1970s: Discovery of entirely new group of organisms (Archaea).
It is true that most archaeans don't look that different from bacteria under the
microscope, and that the extreme conditions under which many species live
has made them difficult to culture, so their unique place among living organisms
long went unrecognized.
However, biochemically and genetically, they are as different from bacteria
as you are. Although many books and articles still refer to them as "Archaebacteria",
that term has been abandoned because they aren't bacteria -- they're Archaea.
In 1965, Linus Pauling and Emile Zuckerland proposed instead using the sequences
of the genes in different prokaryotes to work out how they are related to each other.
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This approach, known as phylogenetics,
is the main method used today.
Dr.Farokh Rokhbakhsh-Zamin
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and Dr.Nadia KAzemiPour

Archaea: Morphology
Archaea are tiny, usually less than one micron long (one one-thousandth of a
millimeter). Even under a high-power light microscope, the largest archaeans
look like tiny dots. Fortunately, the electron microscope can magnify even
these tiny microbes enough to distinguish their physical features.
Much like bacteria, cocci and rods are common shapes, Other shapes can also
exist but no spirochetes or mycelial forms yet. They are branched/flat shapes.
Sizes vary (typically 1-2 x 1-5 m for rods, 1-5 m in diameter for cocci)
Smallest observed is 0.2 m in diameter
Largest is a multicellular form that can reach 30 mm in length!

Archaea for Ph.D. students by


Dr.Farokh Rokhbakhsh-Zamin
and Dr.Nadia KAzemiPour

Archaea: Morphology
Archaea are tiny, usually less than one micron long (one one-thousandth of a
millimeter). Even under a high-power light microscope, the largest archaeans
look like tiny dots. Fortunately, the electron microscope can magnify even these
tiny microbes enough to distinguish their physical features. You can see archaean
images below, made using a variety of micrographic techniques.

Basic Archaeal Shapes :


At far left, Methanococcus janaschii, a coccus form with numerous flagella attached to
one side.
At left center, Methanosarcina barkeri, a lobed coccus form lacking flagella.
At right center, Methanothermus fervidus, a short bacillus form without flagella.
At far right, Methanobacterium thermoautotrophicum, an elongate bacillus form.
Archaea for Ph.D. students by
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Some are spherical, a form known as coccus, and these may be perfectly round
or lobed and lumpy.
Some are rod-shaped, a form known as bacillus, and range from short barshaped rods to long slender hair-like forms.
Some oddball species have been discovered with a triangular shape, or even a
square shape like a postage stamp!
Structural diversity among archaeans is not limited to the overall shape of the
cell. Archaea may have one or more flagella attached to them, or may lack
flagella altogether.
The flagella are hair-like appendages used for moving around, and are attached
directly into the outer membrane of the cell.
When multiple flagella are present, they are usually attached all on one side of
the cell.
Other appendages include protein networks to which the cells may anchor
themselves in large groups.
As with other living things, archaeal cells have an outer cell membrane that
serves as a barrier between the cell and its environment. Within the membrane
is the cytoplasm, where the living functions of the archeon take place and
where the DNA is located.
Archaea for Ph.D. students by
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and Dr.Nadia KAzemiPour

Around the outside of nearly all archaeal cells is a cell wall, a semi-rigid layer
that helps the cell maintain its shape and chemical equilibrium. All three of these
regions may be distinguished in the cells of bacteria and most other living things,
but when you take a closer look at each region, you find that the similarities are
merely structural, not chemical.
In other words, Archaea build the same structures as other organisms, but they
build them from different chemical components.
For instance, the cell walls of all bacteria contain the chemical peptidoglycan.
Archaeal cell walls do not contain this compound, though some species contain a
similar one. Likewise, archaea do not produce walls of cellulose (as do plants) or
chitin (as do fungi). The cell wall of archaeans is chemically distinct.
Like bacteria, archaeans have no internal membranes and their DNA exists as a
single loop called a plasmid.
Their tRNAs have a number of features that differ from all other living things.
The tRNA molecules (short for "transfer RNA") are important in decoding the
message of DNA and in building proteins. Certain features of tRNA structure are
the same in bacteria, plants, animals, fungi, and all known living things -- except
the Archaea. There are even features of archaeal tRNA that are more like
eukaryotic critters than bacteria, meaning that Archaea share certain features in
Archaea
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common with you and not with
bacteria.
Dr.Farokh Rokhbakhsh-Zamin
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Basic Archaeal Structure : The three primary regions of an archaeal cell are
the cytoplasm, cell membrane, and cell wall. Above, these three regions are
labelled, with an enlargement at right of the cell membrane structure.
Archaeal cell membranes are chemically different from all other living things,
including a "backwards" glycerol molecule and isoprene derivatives in place
of fatty acids. See text below for details.
Archaea for Ph.D. students by
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Archeal ribosomes are the giant processing molecules that assemble proteins for
the cell. While bacterial ribosomes are sensitive to certain chemical inhibiting
agents, archaeal and eukaryotic ribosomes are not sensitive to those agents. This
may suggest a close relationship between Archaea and eukaryotes.
The most striking chemical differences between Archaea and other living things
lie in their cell membrane. There are four fundamental differences between the
archaeal membrane and those of all other cells:
(1) chirality of glycerol, (2) ether linkage, (3) isoprenoid chains, and (4)
branching of side chains.
These may sound like complex differences, but a little explanation will make the
differences understandable. The header for each explanation is color-coded to
match the relevant portion of the diagram below.
(1) Chirality of glycerol : The basic unit from which cell membranes are built
is the phospholipid. This is a molecule of glycerol which has a phosphate
added to one end, and two side chains attached at the other end. When the cell
membrane is put together, the glycerol and phosphate end of the molecules hang
out at the surface of the membrane, with the long side chains sandwiched in the
middle (see illustration). This layering creates an effective chemical barrier
around the cell and helps maintain chemical equilibrium.
Archaea for Ph.D. students by
Dr.Farokh Rokhbakhsh-Zamin
and Dr.Nadia KAzemiPour

This is the same situation as the stereoisomers of glycerol. There are two
possible forms of the molecule that are mirror images of each other. It is not
possible to turn one into the other simply by rotating it around.
While bacteria and eukaryotes have D-glycerol in their membranes, archaeans
have L-glycerol in theirs. This is more than a geometric difference. Chemical
components of the cell have to be built by enzymes, and the "handedness"
(chirality) of the molecule is determined by the shape of those enzymes. A cell
that builds one form will not be able to build the other form.

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(2) Ether linkage :


When side chains are added to the glycerol, most organisms bind them together using
an ester linkage (see diagram below).
The side chain that is added has two oxygen atoms attched to one end. One of these
oxygen atoms is used to form the link with the glycerol, and the other protrudes to the
side when the bonding is done.
By contrast, archaeal side chains are bound using an ether linkage, which lacks that
additional protruding oxygen atom. This gives the resulting phospholipid different
chemical proerties from the membrane lipids of other organisms.

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(3) Isoprenoid chains :


The side chains in the phospholipids of bacteria and eukaryotes are fatty acids,
chains of usually 16 to 18 carbon atoms.
Archaea do not use fatty acids to build their membrane phospholipids. Instead,
they have side chains of 20 carbon atoms built from isoprene.
Isoprene is the simplest member of a class of chemicals called terpenes.
By definition, a terpene is any molecule built by connecting isoprene molecules
together, rather like building with Lego blocks. Each isoprene unit has a "head"
and a "tail" end (again like a Lego block), but unlike their toy counterparts,
isoprene blocks can be joined in many ways.
A head can be attached to a tail or to another head end, and tails can be similarly
joined.
The immense variety of terpene compounds that can be built from simple
isoprene units include beta-carotene (a vitamin), natural and synthetic rubbers,
plant essential oils (such as spearmint), and steroid hormones (such as estrogen
and testosterone).

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Archaea for Ph.D. students by


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(4) Branching of side chains :


Not only are the side chains of achaeal membranes built from different components, but
the chains themselves have a different physical structure.
Because isoprene is used to build the side chains, there are side branches off the main
chain (see diagram above). The fatty acids of bacteria and eukaryotes do not have these
side branches (the best they can manage is a slight bend in the middle), and this creates
some interesting properties in archaeal membranes.
For example, the isoprene side chains can be joined together. This can mean that the two
side chains of a single phospholipid can join together, or they can be joined to side chains
of another phospholipid on the other side of the membrane. No other group of
organisms can form such transmembrane phospholipids.
Another interesting property of the side branches is their ability to form carbon rings.
This happens when one of the side branches curls around and bonds with another atom
down the chain to make a ring of five carbon atoms.
Such rings are thought to provide structural stability to the membrane, since they seem
to be more common among species that live at high temperatures.
They may work in the same way that cholesterol does in eukaryotic cells to stabilize
membranes. It's interesting to note that cholesterol is another terpene!

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Composed of unique lipids


isoprene units (five carbon, branched)
ether linkages rather than ester
linkages to glycerol
Some have a monolayer structure
instead of a bilayer structure

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Flexible

Rigid gives
membrane
stability to
thermophiles
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Bacterial cell wall biosynthesis:

Schematic representation of the bacterial cell wall biosynthesis pathway.


Paradis-Bleau et al. BMC BiochemistryArchaea
2008for9:33
doi:10.1186/1471-2091-9-33
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Archaeal Cell Wall:


Lack peptidoglycan

Pseudomurein may be outermost


layer similar to Gram-positive
bacteria.

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Many Gram-negative and Gram-positive bacteria, as well a many archaea possess a


regularly structured layer called an S-layer attached to the outermost portion of their
cell wall. It is composed of protein or glycoprotein and in electron micrographs, has
a pattern resembling a tiled surface. Transmission electron micrograph of a freezeetched, metal shadowed preparation of a bacterial cell with an S-layer with
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hexagonal lattice symmetry. Bar =Dr.Farokh
100nm
.
Rokhbakhsh-Zamin
21
and Dr.Nadia KAzemiPour

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Surface layer structure:


Sequence analyses of S-layer proteins have predicted that they have sizes of 40-200 kDa
and may be composed of multiple domains some of which may be structurally related.
An S-layer is a part of the cell envelope commonly found in bacteria, as well as among
archaea.
It consists of a monomolecular layer composed of identical proteins or glycoproteins. This
structure is built via self-assembly and encloses the whole cell surface. Thus, the S-layer
protein can represent up to 1015% of the whole protein content of a cell.
S-layer proteins are poorly conserved or not conserved at all, and can differ markedly
even between related species. Depending on species, the S-layers have a thickness
between 5 and 25 nm and possess identical pores with 28 nm in diameter.
S-layers exhibit either an oblique (p1, p2), square (p4) or hexagonal (p3, p6) lattice
symmetry. Depending on the lattice symmetry, the S-layer is composed of one (P1), two
(P2), three (P3), four (P4), or six (P6) identical protein subunits, respectively. The centerto-center spacing (or unit cell dimensions) between these subunits range between 2.5 and
35 nm.

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In contrast to single cells, aggregates of Methanosarcina spp. produce a substance called


methanochondroitin covering the S-layer with the latter one also being present in single
cells (Kreisl and Kandler, 1986; Albers and Meyer, 2011). Methanochondroitin, which is
similar to chondroitin in the connecting tissue of vertebrates (Kjellen and Lindahl, 1991),
consists of a repeating trimer of two N-acetylgalactosamines and one glucuronic acid but
differing from vertebral chondroitin in the molar ratio of the monomers and the fact that it is
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not sulfated (Albers and Meyer, 2011).
Dr.Farokh Rokhbakhsh-Zamin
and Dr.Nadia KAzemiPour

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a) Side view of the assembly of surface-layer (S-layer) proteins in different archaea


(Halobacteria145, Sulfolobales145, Thermoproteus spp.145 and Staphylothermus
marinus Natur146; b) Top view of the lattice structure of different S-layers: S-layer
proteins are shown in grey, and pores or holes in white. The red perimeter shows a
single repeating unit in the cases of the p3, p4 and p6 symmetry. Sulfolobales display
p3 symmetry147, Desulphurococcus mobilis displays p4 symmetry148 and
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by N, N-linked glycosylation; O; OThermoproteus tenax displays p6 symmetry149,
150.
Dr.Farokh Rokhbakhsh-Zamin
25
linked glycosylation.
and Dr.Nadia KAzemiPour

a | Schematic representation of a cross-section of the cell envelope of Sulfolobus solfataricus


showing the cytoplasmic membrane, with membrane-spanning tetraether lipids and an Slayer composed of two proteins ? a surface-covering protein (red oval) and a membraneanchoring protein (yellow oblong). b | Schematic representation of a cell envelope of an
archaeon that stains positive with the Gram stain and that contains a pseudomurein layer in
addition to the S-layer. The cytoplasmic
membrane
is composed
of diether lipids.
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Gram + versus Gram - Archaea

Gram positive
Cell wall of pseudomurein or
other complex carbohydrate

Gram negative
No outer membrane
No cell wall
Thick protein/glycoprotein coat

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Ribosomes :
bacterial/archaeal ribosome = 70S but eukaryotic (80S) S = Svedburg unit
Bacterial and archaeal ribosomal RNA:
16S small subunit & 23S and 5S in large subunit
archaea have additional 5.8S (also seen in eukaryotic large subunit)
Proteins vary (archaea more similar to eukarya than to bacteria)
1.

2.

As many Archaea have adapted to function under conditions of extreme


salt or temperature, their ribosomal components are highly resistant to
such adverse conditions, and the overall ribosome structure often has an
higher rigidity than that of mesophilic microorganisms. This makes
archaeal ribosomes optimally suited for crystallographic studies, and in
fact, high-resolution three-dimensional structures have been obtained with
ribosomal crystals from halophilic and thermophilic Archaea.
These studies pioneered the resolution at the atomic level of ribosome
architecture, a feat that won the 2009 Nobel Prize in Chemistry to Ada
Yonath, Thomas Steitz and Venki Ramakrishnan.

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Cannulae:
Members of the genus Pyrodictium were isolated from hydrothermal marine
environments, with growth temperatures ranging from 80 to up to 110C.
hollow, tube like structures on the surface of these archaea
may be involved in formation of networks of multiple daughter cells 25

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Cells grow in a network of tubules termed cannulae, which connect the cells with
each other.
Cannulae are hollow tubes with an outside diameter of 25 nm, and they appear
empty when cross-fractured or thin-sectioned.
They consist of at least three different, but homologous, glycoprotein subunits
with identical N termini but with different molecular masses of 20, 22, and 24
kDa.
The structure of the cannulae is highly resistant to heat as well as denaturing
agents, as no morphological changes were observed after 60 min of incubation at
140C or 10 min at 100C and 2% sodium dodecyl sulfate.
Two newly formed daughter cells always stay connected with the growing
cannulae, leading to a dense network of cells and cannulae at the end of the
cultivation.
The final length of the cannulae varied between 30 and 150 micrometer with an
elongation rate of 1.0 to 1.5 micrometer/min, which is significantly higher than
the elongation rate for bacterial flagella, e.g., Salmonella (0.16 micrometer/min
in in vitro measurements)

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Cannulae function as an intercellular connection between different cells.


Certain marine bacteria produce an unusual tubular surface structure, termed
spinae, that is capable of forming a broadly similar network. Approximately11
nm in diameter and apparently open tubes, these spinae connect cells over
distances of several micrometers.
Spinae might allow exchange of signals between the connected cells.
However, the function of the cannula network is still unclear. It might act to
anchor cells to each other or as a means of communication for the exchange of
either nutrients or even genetic material. Currently, little is known about the
mechanisms and kinetics of diffusion of biological materials within a cannula
tubule.
Part of a network of Pyrodictium
cells and cannulae, with tubules in
a regular array.
Sandy Y. M. Ng et al. J. Bacteriol.
2008;190:6039-6047

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Hami (Hamus):
not well understood
grappling hook appearance
involvement in cell adhesion mechanisms?

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A novel filamentous cell appendage of unexpectedly high complexity.


Archaeal cells bearing these structures are found in macroscopically visible
string-of-pearls-like arrangements among bacterial filaments, mainly Thiothrix
(SM) or IMB1 proteobacterium (IM) growing in cold (10C), sulfidic springs.
The individual pearl can reach a diameter of up to 8 mm. Scanning electron
microscopy and fluorescent in situ hybridization revealed that the filamentous
bacteria form the outer, whitish part of the pearl, as well as the connecting
threads, with each interior of the pearl dominated by up to 107 euryarchaeal
SM1 cells.
The archaeal cells are coccoids approximately 0.6 micrometer in diameter.
Attached to each archaeal cell are approximately 100 filamentous hami, each 1
to 3 micrometer in length and 7 to 8 nm in diameter (Fig. next page, a).
The hamus filament has a helical basic structure, with three prickles (each 4 nm
in diameter) emanating from the filament at periodic distances (46 nm). At the
distal end, a tripartite, barbed grappling hook, 60 nm in diameter, was identified
(Fig. 5b and c). The hamus is composed mainly of a 120-kDa protein.
Chemical testing revealed that the hami remain stable over broad temperature
and pH ranges (0 to 70C; pH 0.5 to 11.5). As such, these filamentous structures
mediate strong cellular adhesion for the archaeal cells to surfaces of different
chemical compositions.
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FIG. (a) Electron micrograph of a platinumshadowed SM1 Euryarchaeal coccus. About


100 hami emanate radially from the cell
surface. (b) Electron micrographs of grappling
hooks located at the distal ends of the hami.
Arrowheads indicate locations of barbs. (c)
Electron micrograph of high-level-structured
SM1 hami. The hami show prickles and
grappling hooks.
(Reprinted from reference 52 with permission
of the publisher.)

Sandy Y. M. Ng et al. J. Bacteriol. 2008;190:6039-6047


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Pili:
Sulfolobus cells taken freshly from a hot spring are attached to sulfur particles
by numerous 5-nm wide pili. Pili have also been observed in many other
archaea.
There are glycosylation pathway to the assembly of both flagella and pili in
Methanococcus maripaludis. Deletion of an acetyltransferase gene involved in
the biosynthesis of the glycan that is N linked to the flagellins resulted in
nonflagellated cells. Unexpectedly, this mutant also lacked pili on the cell
surface. Pili were found in the culture supernatant, indicating that deletion of the
gene did not affect pilus formation but rather affected attachment of the pili to
the cell surface.
Even though the pilins had similarities to bacterial type IV pilins, the structure
formed by the archaeal pilins is unlike that bacterial pili.
Investigation of the biochemistry, genetics, and functions of archaeal pili has
only recently begun. Consequently, it is presently unclear if archaea possess the
vast diversity of pilus types, with assorted functions and assembly mechanisms,
presently known to occur in bacteria.

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Methanococcus maripaludis showing presence of flagella and thinner pili (arrows).

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Sandy Y. M. Ng et al. J. Bacteriol. 2008;190:6039-6047
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FIG. 6. Pili on the surface of a flaK mutant of Methanococcus maripaludis. This mutant
cannot make flagella, leaving the peritrichously located pili evident as the only surface
structure remaining on the cell surface. The sample was negatively stained with 2%
phosphotungstic acid, pH 7. Bar, 200
nm.
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Courtesy of S.-I. Aizawa, reproducedDr.Farokh
with permission.
Rokhbakhsh-Zamin
37
and Dr.Nadia KAzemiPour

Bindosome:
The bindosome in Sulfolobus solfataricus.
the actual structure has not been visualized in the membranes but rather it is
thought to be a pilus-like structure close to the cytoplasmic membrane or
integrated within the S-layer.
The main evidence in support of the presence of this hypothesized structure is
that the proposed structural components, the substrate binding proteins (SBPs),
contain class III signal peptide sequences, a feature typical of proteins which are
well known to form oligomeric structures in both archaea and bacteria.
The oligomerized complex is proposed to play a role in facilitating sugar uptake,
a function that enables Sulfolobus solfataricus to grow on a broad variety of
substrates.
Sulfolobus species are hyperthermophilic acidophiles typically found in volcanic
springs, with optimal growth at around pH 2 to 3 in the temperature range of 75
to 80C.
One interesting distinction that draws S. solfataricus apart from other Sulfolobus
species, such as S. tokodaii and S. acidocaldarius, is the ability to grow on a
wide variety of sugars as its only carbon source.
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Previous studies showed that S. solfataricus has a wide range of ABC transport systems
for sugar uptake. ABC transporters encompass the actual transport domain in the
membrane and cytoplasmically located ATPases, which drive the transport of the
substrate by ATP hydrolysis
At the periplasmic side of the membrane, SBPs bind the substrate and deliver it to the
transport domain. Interestingly, the signal peptides from one class of sugar binding
proteins of S. solfataricus resemble class III signal peptides found in archaeal flagellins or
bacterial pilins.
The controlled assembly/surface localization of SBPs probably enables the organism to
scavenge nutrients more readily from the environment to concentrate substrates in the
periplasmic space between the S-layer and the cytoplasmic membrane.
The bindosome may represent an affinity cascade that channels substrates to the ABC
transporters. Whether the SBPs are assembled into a pilus-like structure or are attached
in a controlled manner to the S-layer remains to be determined(Fig. 7).

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FIG. 7. (A) Model of the assembly of surface structures in the cell envelope of Sulfolobus
solfataricus: Precursor proteins (SBPs, prepilins, or preflagellins) are processed by PibD and are
then inserted by their specific assembly system either in the bindosome structure, the UV
inducible pili or the flagellum. The exact nature of the bindosome structure is not known yet,
and an alternate format, shown attached to the S-layer, is also indicated. All three assembly
systems share the same core of the machinery: an integral membrane protein and a
cytoplasmic ATPase. (B) Electron micrograph
of flagellated S. solfataricus P2 cells. Flagella are
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present all around the cells and do notDr.Farokh
appearRokhbakhsh-Zamin
bundled. Bar, 1 m.
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Archaea
Similarities to prokaryotes
Size
Shape
Lack nucleus
Single chromosome
Genes in operons
No introns
Similarities to eukaryotes
Few plasmids
RNA polymerase/promoters
Translation machinery: ribosome and tRNA
Sequencing of Methanococcus jannaschii in 1992 showed 56% of genes not
similar to bacteria or eukaryotes!

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Differences and similarities between Flagella of Archaea &


Bacteria:

Archaeal Flagella are thinner


More than one type of flagellin protein
Flagella are not hollow
assembly of archaeal flagella might occur with subunits added at the base Hook
and basal body
Growth occurs at the base, not the end
Archaeal flagella are more similar to bacterial type IV pili than to bacterial
flagella
Both rotations are the same
Archaea do not possess any homologues of genes found in bacteria that are
involved with bacterial flagellum structure or assembly
They have a similar chemotaxis system as found in bacteria under controlling by
che genes
Simillar rotation as bacteria with rotation switching
Archaeal flagella used for (swimming, connecting cells perhaps as an initial
prerequisite for genetic transfer, in adhesion to abiotic surfaces).
it has been shown that interactions between Pyrococcus furiosus and
Methanopyrus kandleri can occur
flagella
as well as cell-to-cell contact,
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Rokhbakhsh-Zamin
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resulting in the formation of aDr.Farokh
structured
bispecies
biofilm.
and Dr.Nadia KAzemiPour

Proposed model for assembly of archaeal flagella using Methanococcus voltae as a model.
Sandy Y. M. Ng et al. J. Bacteriol. 2008;190:6039-6047
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The Nucleoid
Irregularly shaped region in bacteria and archaea
Usually not membrane bound (few exceptions)
Location of chromosome and associated proteins
Usually 1 (some evidence for polyploidy in some archaeons)
Supercoiling and nucleoid proteins (histones, Alba, condensins) aid in folding

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Bacteriorhodopsin:
The halophilic archaeon makes use of light for both energy and sensory
transduction by exploiting a family of light-sensitive proteins. The archaeal
rhodopsin, a 26.5 kDa protein, has a transmembrane domain of seven helical
protein segments which photons are captured via a retinal chromophore. The
excellent thermodynamic and photochemical stability of bacteriorhodopsin has
led to many uses in technical applications like holography, spatial light
modulators, artificial retina, neural network optical computing, and volumetric
and associative optical memories.

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Archaea systematics:
Reliable and repeatable classification of bacteria was not possible until the late 20th
century when molecular biology made it possible to sequence their DNA.
Because ribosomes are so critically important is the functioning of living things, they are
not prone to rapid evolution.
A major change in ribosome sequence can render the ribosome unable to fulfill its
duties of building new proteins for the cell. Because of this, we say that the sequence in
the ribosomes is conserved -- that it does not change much over time. This slow rate of
molecular evolution made the ribosome sequence a good choice for unlocking the
secrets of bacterial evolution. By comparing the slight differences in ribosome sequence
among a wide diversity of bacteria, groups of similar sequences could be found and
recognized as a related group.

In the 1970s, Carl Woese and his colleagues at the University of Illinois at UrbanaChampaign began investigating the sequences of bacteria with the goal of developing a
better picture of bacterial relationships. Their findings were published in 1977, and
included a big surprise. Not all tiny microbes were closely related. In addition to the
bacteria and eukaryote groups in the analysis, there was a third group of methaneproducing microbes. These methanogens were already known to be chemical oddities in
the microbial world, since they were killed by oxygen, produced unusual enzymes, and
had cell walls different from all known bacteria.
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These bacteria that lived at high temp. or produced methane clustered together
as a group well away from the usual bacteria and the eubacteria.
Because of this vast difference in genetic makeup, Woese proposed that life be
divided into three domains: Eukaryota, Eubacteria, and Archaebacteria.
He later decided that the term Archaebacteria was a misnomer, and shortened it
to Archaea. The three domains are shown in the next page, which illustrates
also that each group is very different from the others.

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Archaeans include inhabitants of some of the most extreme environments on the

planet.
Some live near rift vents in the deep sea at temperatures well over 100 degrees
Centigrade.
Others live in hot springs (such as the ones pictured above next page), or in
extremely alkaline or acid waters.
They have been found thriving inside the digestive tracts of cows, termites,
and marine life where they produce methane.
They live in the anoxic muds of marshes and at the bottom of the ocean, and
even thrive in petroleum deposits deep underground.
Some archaeans can survive the dessicating effects of extremely saline
waters. One salt-loving group of archaea includes Halobacterium, a wellstudied archaean. The light-sensitive pigment bacteriorhodopsin gives
Halobacterium its color and provides it with chemical energy.
Bacteriorhodopsin has a lovely purple color and it pumps protons to the
outside of the membrane. When these protons flow back, they are used in the
synthesis of ATP, which is the energy source of the cell. This protein is
chemically very similar to the light-detecting pigment rhodopsin, found in the
vertebrate retina.
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Finding Archaea : The hot springs of Yellowstone


National Park, USA, were among the first places
Archaea were discovered. At above is Octopus Spring,
and at dawn is Obsidian Pool. Each pool has slightly
different mineral content, temperature, salinity, etc., so
different pools may contain different communities of
archaeans and other microbes. The biologists pictured
above are immersing microscope slides in the boiling
pool onto which some archaeans might be captured for
study.
Archaeans may be the only organisms that can live in extreme habitats such
as thermal vents or hypersaline water.
They may be extremely abundant in environments that are hostile to all other
life forms.
However, archaeans are not restricted to extreme environments; new research
is showing that archaeans are also quite abundant in the plankton of the open
sea. Much is still to be learned about these microbes, but it is clear that the
Archaea for Ph.D. students by
Archaea is a remarkably diverse
and successful clade of organisms.
Dr.Farokh Rokhbakhsh-Zamin
50
and Dr.Nadia KAzemiPour

Comparison of Archaea to the other domains:


The following table compares some major characteristics of the three domains, to illustrate their
similarities and differences. Many of these characteristics are also discussed below.
Property

Archaea

Bacteria
Ester-linked lipids,
peptidoglycan

Eukarya

Cell Membrane

Ether-linked lipids,
pseudopeptidoglycan

Gene Structure

Circular chromosomes, similar Circular chromosomes,


translation and transcription to unique translation and
Eukarya
transcription

Multiple, linear
chromosomes, similar
translation and
transcription to Archaea

Internal Cell
Structure

No membrane-bound
organelles or nucleus

No membrane-bound
organelles or nucleus

Membrane-bound
organelles and nucleus

Metabolism

Various, with methanogenesis


unique to Archaea

Various, including
photosynthesis, aerobic and
Photosynthesis and
anaerobic respiration,
cellular respiration
fermentation, and autotrophy

Reproduction

Asexual reproduction,
horizontal gene transfer

Asexual reproduction,
horizontal gene transfer

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Ester-linked lipids,
various structures

Sexual and asexual


reproduction

51

Archaean Phylogeny :
The phylogeny of archaeans is based on
molecular sequences in their DNA. The
analysis of these sequences reveals three
distinct groups within the Archaea.
1. The Euryarcheota are probably the best
known, including many methaneproducers and salt-loving archaeans.
2. The Crenarcheota include those
species that live at the highest
temperatures of any known living things,
though a wide variety have recently been
discovered growing in soil and water at
more moderate temperatures.
3. The Korarcheota are only known from
their DNA sequences -- nothing more is
known about them yet since they have
only recently been discovered.
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In many ways, archaeal cells resemble the cells of bacteria, but in a number of important
respects, they are more like the cells of eukaryotes. The question arises whether the
Archaea are closer relatives of the bacteria or our our group, the eukaryotes.
This is a very difficult question to answer, because we are talking about the deepest
branches of the tree of life itself; we do not have any early ancestors of life around today
for comparison.
One novel approach used in addressing the question is to look at sequences of
duplicated genes. Some DNA sequences occur in more than one copy within each cell,
presumably because an extra copy was made at some point in the past. There are a very
few genes known to exist in duplicate copies in all living cells, suggesting that the
duplication happened before the separation of the three domains of life.
In comparing the two sets of sequences, scientists have found that the Archaea may
actually be more closely related to us (and the other eukaryotes) than to the bacteria.

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Major groups of Archaea


1.
2.
3.
4.
5.

Methanogenic archaea
Archaeal sulfate reducers
Extremely halophilic archaea
Cell wall-less archaea
Extremely thermophilic S0-metabolizers

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Methanogens:
1.
2.
3.
4.
5.
6.

Largest group of Archaea


Form CH4 from CO2 or other compounds (e.g. formate, methanol, acetate)
Strict anaerobes
Found in a variety of anaerobic environments rich in organic matter
Causes cows to belch!
Methane: energy source vs. greenhouse gases

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Cell wall-less Archaea:


Thermoplasma species (T. acidophilum, T. Volcanicum) ; Picrophilaceae
Characteristics:
1. Staining: negatively in the Gram reaction
2. Morphology: coccoid cell
3. Motility may be motile and flagellated
4. Specialized structures coccoid cells lacking a cell envelope
5. Facultative anaerobic
6. Obligate acidophile
7. Obligate thermophile grows at 33-67 oC
8. Require yeast extract for growth
9. Cytoplasmic membrane contains a mannose rich glycoprotein and a lipoglycan
10. RNA polymerase of the BAC type
11. Membrane contains ether lipids characteristic of archaea
12. G+C Mol % ( T. Acidophilum 46 / T. volacanicum 38)
13. Warm, acidic environment
14. Thermoplasma isolated from coal refuse piles 55-59C, pH 1-2
15. Picrophilaceae can grow at pH=0
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Extremely Thermophilic Archaea:

1.
2.
3.
4.
5.
6.
7.
8.

(Acidianus, Desulfurococcus, Desulfurolobus ambivalens, Hyperthermus butylicus,


Metallosphaera sedula, Pyrobaculum, Pyrococcus, Pyrodictium, Sothermus
marinus, Sulfolobus, Thermococcus, Thermodiscus maritimus, Thermofilum,
Thermoproteus)
Gram negative rods, filaments or cocci
aerobic, facultatively anaerobic but mainly strictly anaerobic
Acidophiles and neutrophiles
Obligately thermophilic 70-105 C Optimal growth
between 70 and 105 C.
autotrophic or heterotrophic growth
Most species are sulfur metabolizers
S0 reduce to sulfide
Hot springs of yellow stones

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Halophilic Archaea:
(Eubacteria, Haloarcula, Halobacterium, Halococcus, Haloferax, Natronobacterium,
Natronococcus occultus)

1.
2.
3.
4.
5.
6.
7.
8.

Gram negative or Gram positive, Rods and regular to highly irregular cells.
chemooorganotrophs
aerobic or facultatively anaerobic
Neutrophilic or alkalophilic.
Mesophilic or slightly thermophilic (up to 55 C).
requirement for high concentrations of sodium chloride (1.5 Molar or above).
Carotenoids give reddish color
Bacteriorhodopsin capture light for energy in anaerobic respiration

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Sulfate reducing Archaea:

Archaeoglobus (A. fulgidus A. profundus)


Blue green fluorescence at 420 nm under the UV microscope
strictly anaerobic
Extremely thermophilic (growth up to 92oC, optimum 83oC).
reduce sulfate to form hydrogen sulfide, Produce traces of methane
Isolated from deep sea thermal vent

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Methanogenes:
Subgroup 1:
Methanobacterium, Methanobrevibacter, Methanosphaera, Metthanothermus
Subgroup 2 :
Methanococcus, Methanocorpusculum, Methanoculleus, Methanogenium
Methanolacinia paynteri, Methanomicrobium mobile, Methanoplanus,
Methanospirillum hungatei
Subgroup 3:
Methanococcoides methylutens, Methanohalobium evstigatus, Methanohalophilus,
Methanolobus, Methanosarcina, Methanothrix
1. Gram-negative or Gram-positive
2. A wide range of morphological types Cells are coccoid bodies, pseudosarcina or
rods
3. nonmotile or motile
4. Very strictly anaerobic
5. Produces methane
6. Cells do not contain muramic acid. Lipids are predominantly isoprenoid
hydrocarbons ether-linked to glycerol.
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7.

The methonagens which are also called Methanomicrobiales form a highly


specialized physiological group which does not utilize carbohydrate,
proteinaceous materials or other organic compounds as energy sources other
than those listed. They grow by oxidation of H2 or formate with the reduction
of carbon dioxide to methane or by fermentation of methylated amines,
methanol or acetate to methane and carbon dioxide. Some strains oxidize 1propanol, 2-propanol, 2-butanol or ethanol to propionate, acetone, 2-butanone
or acetate(respectively) and reduce carbon dioxide to methane.
8. They are widely distributed in nature, being found in anaerobic habitats, such as
aquatic sediments, anaerobic sewage-sludge digesters and the gastrointestinal
tracts of animals.

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Biotechnological Applications:
The extremophilic nature of many Archaea: to probe the potential
biotechnological applications of their stable cellular components.
This is particularly true of their enzymes (called extremozymes), which are
able to remain catalytically active under extremes of temperature, salinity, pH
and pressure.
Many interesting enzymes have been isolated from extremophilic microbes.
Specific archaeal metabolites have also been purified and characterized and
some of them have potential industrial uses.
Genes encoding several enzymes from extremophiles have been cloned in
mesophilic hosts, with the objective of overproducing the enzyme and altering
its properties to suit commercial applications.
Escherichia coli, Bacillus subtilis and yeasts have been used successfully as
mesophilic hosts for several archaeal genes. Genetic engineering techniques are
valuable tools for creating novel biocatalysts that can improve bioprocesses and
facilitate the realization of innovative or novel biotransformations.
Molecular biology has the potential both to overcome the limitations on
enzyme availability and to design new biocatalysts specific to a particular
industrial purposes.
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Table 1. Industrial applications of archaeal products.


Phenotype

Condition

Thermophile

High temp. (45 - 110C) Amylase

Psychrophile

Product

Low temp. (>15C)

Application
Glucose, fructose for sweeteners

Xylanases

Paper bleaching

proteases

Baking, brewing, detergents

DNA polymerase

Genetic engineering

Proteases

Cheese maturation, dairy


production

Dehydrogenases

Biosensors

Amylases

Polymer degradation in detergents

Acidophile

Low pH (0-4)

Sulfor oxidation

Desulfurization of coal

Alkalophile

High pH (8-11)

Cellulase

Polymer degradation in detergents

Halophile

High salt concentration

Whole M.O.

Biopolymer

Piezophile

High pressure

Whole M.O.

Formation of gels and starch


granules

Methalophile High metal concentration

Whole M.O.

Bioremediation, biomineralization

Radiophile

Whole M.O.

Bioremediation of radionuclide
contaminated sites

High radiation level

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Gluco hydrolyzing enzymes:


Complex structure of starch so, a number of enzymes are needed for its
degradation.
Endo-acting enzymes, such as -amylase, hydrolyse linkages in the interior of
the starch polymer in a random fashion, which leads to the formation of linear
and branched oligosaccharides.
Exo-acting enzymes ( and -glucoamylases) attack the substrate from the
nonreducing end, producing oligo and/or monosaccharides.
The finding of new extremely thermostable starch-hydrolysing enzymes, such as
amylases and pullulanases, will significantly improve the industrial starch
bioconversion process.
Investigations concerning recombinant -amylases from Pyrococcus woesei
indicate their suitability for starch processing.
The study of recombinant -galactosidase from P. woesei suitable for purpose of
low lactose milk and whey production is also presented.
The activity of this enzyme in a wide pH range of 4.3-6.6 and high
thermostability suggests that it can be used for processing of dairy products at
temperatures which restrict microbial growth during a long operating time of
continuous-flow reactor with an immobilized enzyme system.
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Preparation of recombinant -amylase and -galactosidase was facilitated by


cloning and expression of genes from P. woesei in E. coli host. Satisfactory level
of recombinant enzymes purification was achieved by thermal denaturation and
precipitation of native proteins originated from E. coli. The obtained -amylase
has maximal activity at pH 5.6 and 93C. The half-life of this preparation (pH
5.6) at 90C and 110C was 11 h and 3.5 h, respectively.
A hyperthermostable glycosidase enzyme with pullulanase activity at 90C from
Thermococcus aggregans was cloned and expressed in E. coli. Unlike all other
pullulan-hydrolyzing enzymes described, the enzyme is able to attack -1,6- as
well as -1,4-glycosidic linkages, affording a mixture of maltotriose, maltose and
glucose. The enzyme is also able to degrade starch, amylose and amylopectin,
forming maltotriose and maltose as main products.
Halophilic glycosidases have also been identified, including a halophilic galactosidase from Haloferax alicantei. The extremely halophilic galactosidase
from Haloferax alicantei is optimally active at 4 M NaCl. Purification of the
enzyme was facilitated by the ability of sorbitol to stabilize enzyme activity in the
absence of salt, which allowed conventional ion-exchange chromatography.

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The continuous production of halophilic -amylase can be performed via wholecell immobilization of Halobacterium salinarum in alginate beads and a
polyvinyl alcohol film. The cells were osmotically stable and showed
continuous enzyme production for 45 days. The stabilized cells could be
permeabilized by chloroform treatment without leakage of the intracellular
components. Using this procedure, cells can be reused under improved
stabilized conditions for biotechnological applications

DNA processing enzymes:


One of the most important advances in molecular biology was the development
of the polymerase chain reaction (PCR). Thermostable DNA polymerases play
a major role in PCR and in a variety of molecular biological applications, e.g.
DNA amplification, cloning, sequencing or labeling. Several native and
recombinant polymerases have been purified and characterized.
DNA polymerase I from the bacterium Thermus aquaticus, called Taq
polymerase, was the first thermostable DNA polymerases utilized in PCR.
Archaeal proof-reading polymerases, such as Pwo from Pyrococcus woesei,
Pfu from P. furiosus, Deep Vent polymerase from the Pyrococcus strain GB-D
and Vent polymerase from Thermococcus litoralis, have an error rate that is up
to tenfold lower than that of Taq
polymerase.
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Esterases and lipases:


In the field of biotechnology, esterases are receiving increasing attention
because of their application in organic biosynthesis.
In aqueous solution, esterases catalyze the hydrolytic cleavage of esters to form
the constituent acid and alcohol whereas, in organic solutions, the
transesterification reaction is promoted.
Both the reactants and the products of transesterification are usually highly
soluble in the organic phase and the reactants may even form the organic phase
themselves.
The P. furiosus esterase and lipase genes have been cloned in E. coli and the
functional properties have been determined.
The archaeal enzyme is the most thermostable (a half-life of 50 min at 126C)
and thermoactive (optimum temperature of 100C) esterase known to date.

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Archaeosomes:
Liposomes are lipid-bilayer bounded vesicles that can be used as a delivery vehicle
for certain vaccines, enzymes and drugs. The term Archaeosome was
introduced by Sprott and co-workers to describe liposomes made with ether lipids
that are unique to the Archaea domain confering considerable stability on liposomal
vesicles. Extensive mouse model studies involving intravenous, oral and
subcutaneous administration of archaeosomes demonstrated that archaeosomes are
safety molecules and they are not toxic.

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Proteases and Peptidases:


The amount of proteolytic enzymes produced world-wide on a commercial scale
is larger than that of any of the other biotechnologically used enzymes.
Serine alkaline proteases are used as additives to household detergents for
laundering where they must resist denaturation by detergents and alkaline
conditions.
The leather industry uses proteinases with high keratinolytic and elastolytic
activities for soaking. The discovery of proteases that can catalyze reactions
under extreme conditions (high temperatures and extremes of pH) will be
valuable for industrial applications.
An extracellular serine protease from the extreme halophile Halobacterium
halobium may be an excellent catalyst for peptide synthesis, exploiting their
reverse reaction, particularly for glycine-containing peptides. The enzyme
requires 4 M NaCl for optimal catalytic activity and stability in aqueous
solutions. The stabilization of halophilic enzymes by organic solvents while
lowering the required salt concentration is of practical importance because high
NaCl concentrations can be corrosive to metals. Higher substrate solubility in
the presence of an organic solvent can be useful in synthetic reactions catalyzed
by enzymes from extreme halophiles.
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Many biotechnologically interesting serine proteases have been identified and


characterized.
Among them is a cell associated serine protease, characterized from
Desulfurococcus strain SY, which showed a half-life of 4.3 h at 95C. A globular
serine protease from Staphylothermus marinus was found to be extremely
thermostable. This enzyme has residual activity even after 10 min of incubation
at 135C.
Another gene encoding a subtilisin-like serine protease, named aereolysin, has
been cloned from Pyrobaculum aerophilum and the protein was modelled on the
basis of structures of subtilisin-type proteases.
Multiple proteolytic activities have been observed in Pyrococcus furiosus. The
cell envelope associated serine protease of P. furiosus, called pyrolysin, was
found to be highly stable with a half-life of 20 min at 105C. The pyrolysin gene
was cloned and sequenced and it was shown that this enzyme is a subtilisin-like
serine protease.
Proteases have also been characterized from the thermoacidophilic archaeon,
Sulfolobus solfataricus and Sulfolobus acidocaldarius.

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In addition to the serine proteases, other types of enzymes have been identified
in extremophiles. Good examples are a thiol protease (propylpeptidase) from
Pyrococcus sp. and a new type of protease from P. furiosus.
Psychrophilic enzymes have potential applications in a broad range of
industrial, agricultural and medical processes. The recent interest in this field is
due to the challenge of finding stable proteases that function in cold water.
In addition, peptidyl synthesis studies with mesophilic enzymes have shown
that low temperature favors high yields because of reduced hydrolysis of the
acyl-enzyme intermediate.
In most other processes, reduced energy consumption due to low temperature
operation will be a significant advantage.

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