Therefore, it was very difficult to isolate them from permanently cold environments. Isolation itself is not a difficult
process, but it requires some precautions: the cold chain
must not be interrupted, which means that all sampling and
laboratory operations (spreading, culturing, isolation, etc.)
must be realized at temperatures below 10
C. Similarly, all
the laboratory equipment (pipettes, culture media, flasks,
etc.) must be kept permanently cold, before and during use.
Psychrophilic bacteria have temperature optima between
8 and 15
C and are generally inhibited and killed over
15–20
C. Their marked sensitivity at room temperature
(quick death) was such that many scientists have long sought
to isolate them without success, and thus, these prokaryotes
have been little studied. During handling, psychrotrophs
were selected during the warming period and were considered as more abundant. But in permanently cold
environments, strict psychrophiles are likely abundant.
Today, they are increasingly isolated thanks to the more
stringent technical control of the temperature. Microeukaryotes that continuously live in cold environments are
qualified of cryophilic microorganisms.
The minimum temperature of psychrophilic bacteria
development is difficult to identify because it is often
in the freezing zone. Formation of colonies has often
been observed at À11
C on agar surfaces. In the cell,
biosynthesis stops at À30
C and the limit of biochemical
reactions (tested in liquid with glycerol as antifreeze added)
was found to be at À140
C. Glycerol penetrates into cells
and protects them by preventing the freezing of water
molecules.
Growth maximum occurs around 6–10
C, sometimes
15
C (Fig. 9.21b). One to 2
C above it, the syntheses of
macromolecules (DNA, RNA, proteins) stop, and 5
C
above, destruction of cellular material occurs with a leak of
macromolecules through membranes such as amino acids
and enzymes, resulting in cell death.
In psychrophilic bacteria, different abnormally thermolabile enzymes have been identified. These enzymes which
exhibit psychrophiles traits have optimum of activity at
15–20
C and are inhibited and denatured beyond 25
C.
However, if there are some unusually labile enzymes in a
psychrophilic bacterium, other enzymes are normally resistant to heat. In addition, some enzymes are thermolabile in a
psychrophile and not thermolabile in another. Common
enzymes presenting psychrophilic characters are malate
dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, hexokinase, aldolase, and phosphoglucose
isomerase.
These enzymes are adapted to low temperatures due to
their structure being different from their mesophilic
homologs: they contain more polar amino acids and fewer
and fewer weak interactions between their different
domains, thus promoting flexibility at low temperature.
Their secondary structure comprises a higher proportion of
helices and less sheets, thus contributing to a reduced rigidity at low temperature.
Psychrophiles also possess membranes containing a high
proportion of unsaturated fatty acids with several double
bonds (up to 4 or 5) for a better functionality and fluidity
of the cytoplasmic membrane at low temperature, whereas in
the mesophilic bacteria, the membrane which contains more
saturated fatty acids becomes waxy, rigid, and nonfunctional
at low temperature. The high proportion of unsaturated fatty
acids in psychrophilic prokaryotes causes a fragility of the
membrane at ambient temperature and thus favors leakage
of macromolecules through the membrane and cell death.
An inability to acylate tRNA at room temperature was also
observed in psychrophilic prokaryotes, whereas acylation
occurs at low temperature. All these peculiarities allow
adaptation to low temperatures, but result in high brittleness
at room temperature.
9.6.2 Oxidative Stress
Molecular oxygen, O 2 , possesses two electrons in its outer
orbitals. It presents a high redox potential and is a powerful
oxidizing agent. The two electrons are separated on two
outer orbitals, and thus oxygen is not directly reactive and
needs to be activated. The inactive state is called triplet
oxygen. The high-energy form is called singlet oxygen.
This is the most reactive and more toxic with a super-oxidant
power; it is also the most widespread in the living world,
produced in the cells of aerobic organisms. This form is also
chemically produced and present in the air, especially in
mists. In cells, singlet oxygen is produced by different
enzymes (myeloperoxidases, photosynthetic systems) and
during photooxidation reactions involving photosensitive
molecules (pigment molecules); its superactive state can
be unwanted and can be destroyed by oxidation vital
compounds in cells. Organisms that are in contact with
this compound possess molecules, such as carotenoids,
which can deactivate this compound by transforming it in
triplet oxygen.
During aerobic respiration, triplet oxygen is reduced
to H 2 O:
O 2 þ 4e
À
þ 4H
þ
! 2H 2 O
This reduction which requires four electrons occurs in
four stages and produces four toxic forms of oxygen:
1. O 2 + e
À
! O 2
À (superoxide ion)!HO 2
• (peroxide radical)
2. O 2
À + e
À + 2 H
+
! H 2 O 2 (hydrogen peroxide)
3. H 2 O 2 + e
À + H
+
! H 2 O + OH
• (hydroxyl radical)
4. OH
• + H
+ + e
À
! H 2 O
The superoxide ion and radical peroxide are formed in
small quantities during respiration and are also produced by
photooxidation in the atmosphere. In cells, they are
9 Adaptations of Prokaryotes to Their Biotopes and to Physicochemical Conditions. . .
327
process, but it requires some precautions: the cold chain
must not be interrupted, which means that all sampling and
laboratory operations (spreading, culturing, isolation, etc.)
must be realized at temperatures below 10
C. Similarly, all
the laboratory equipment (pipettes, culture media, flasks,
etc.) must be kept permanently cold, before and during use.
Psychrophilic bacteria have temperature optima between
8 and 15
C and are generally inhibited and killed over
15–20
C. Their marked sensitivity at room temperature
(quick death) was such that many scientists have long sought
to isolate them without success, and thus, these prokaryotes
have been little studied. During handling, psychrotrophs
were selected during the warming period and were considered as more abundant. But in permanently cold
environments, strict psychrophiles are likely abundant.
Today, they are increasingly isolated thanks to the more
stringent technical control of the temperature. Microeukaryotes that continuously live in cold environments are
qualified of cryophilic microorganisms.
The minimum temperature of psychrophilic bacteria
development is difficult to identify because it is often
in the freezing zone. Formation of colonies has often
been observed at À11
C on agar surfaces. In the cell,
biosynthesis stops at À30
C and the limit of biochemical
reactions (tested in liquid with glycerol as antifreeze added)
was found to be at À140
C. Glycerol penetrates into cells
and protects them by preventing the freezing of water
molecules.
Growth maximum occurs around 6–10
C, sometimes
15
C (Fig. 9.21b). One to 2
C above it, the syntheses of
macromolecules (DNA, RNA, proteins) stop, and 5
C
above, destruction of cellular material occurs with a leak of
macromolecules through membranes such as amino acids
and enzymes, resulting in cell death.
In psychrophilic bacteria, different abnormally thermolabile enzymes have been identified. These enzymes which
exhibit psychrophiles traits have optimum of activity at
15–20
C and are inhibited and denatured beyond 25
C.
However, if there are some unusually labile enzymes in a
psychrophilic bacterium, other enzymes are normally resistant to heat. In addition, some enzymes are thermolabile in a
psychrophile and not thermolabile in another. Common
enzymes presenting psychrophilic characters are malate
dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, hexokinase, aldolase, and phosphoglucose
isomerase.
These enzymes are adapted to low temperatures due to
their structure being different from their mesophilic
homologs: they contain more polar amino acids and fewer
and fewer weak interactions between their different
domains, thus promoting flexibility at low temperature.
Their secondary structure comprises a higher proportion of
helices and less sheets, thus contributing to a reduced rigidity at low temperature.
Psychrophiles also possess membranes containing a high
proportion of unsaturated fatty acids with several double
bonds (up to 4 or 5) for a better functionality and fluidity
of the cytoplasmic membrane at low temperature, whereas in
the mesophilic bacteria, the membrane which contains more
saturated fatty acids becomes waxy, rigid, and nonfunctional
at low temperature. The high proportion of unsaturated fatty
acids in psychrophilic prokaryotes causes a fragility of the
membrane at ambient temperature and thus favors leakage
of macromolecules through the membrane and cell death.
An inability to acylate tRNA at room temperature was also
observed in psychrophilic prokaryotes, whereas acylation
occurs at low temperature. All these peculiarities allow
adaptation to low temperatures, but result in high brittleness
at room temperature.
9.6.2 Oxidative Stress
Molecular oxygen, O 2 , possesses two electrons in its outer
orbitals. It presents a high redox potential and is a powerful
oxidizing agent. The two electrons are separated on two
outer orbitals, and thus oxygen is not directly reactive and
needs to be activated. The inactive state is called triplet
oxygen. The high-energy form is called singlet oxygen.
This is the most reactive and more toxic with a super-oxidant
power; it is also the most widespread in the living world,
produced in the cells of aerobic organisms. This form is also
chemically produced and present in the air, especially in
mists. In cells, singlet oxygen is produced by different
enzymes (myeloperoxidases, photosynthetic systems) and
during photooxidation reactions involving photosensitive
molecules (pigment molecules); its superactive state can
be unwanted and can be destroyed by oxidation vital
compounds in cells. Organisms that are in contact with
this compound possess molecules, such as carotenoids,
which can deactivate this compound by transforming it in
triplet oxygen.
During aerobic respiration, triplet oxygen is reduced
to H 2 O:
O 2 þ 4e
À
þ 4H
þ
! 2H 2 O
This reduction which requires four electrons occurs in
four stages and produces four toxic forms of oxygen:
1. O 2 + e
À
! O 2
À (superoxide ion)!HO 2
• (peroxide radical)
2. O 2
À + e
À + 2 H
+
! H 2 O 2 (hydrogen peroxide)
3. H 2 O 2 + e
À + H
+
! H 2 O + OH
• (hydroxyl radical)
4. OH
• + H
+ + e
À
! H 2 O
The superoxide ion and radical peroxide are formed in
small quantities during respiration and are also produced by
photooxidation in the atmosphere. In cells, they are
9 Adaptations of Prokaryotes to Their Biotopes and to Physicochemical Conditions. . .
327