Determination of Microbial Production
203
above-listed basic assumptions proved to be either not correct or completely
wrong (Karl 1986; Robarts and Zohary 1993). In any case, it is sensible to
salvage something from this method that can be of practical use, in view of the
fact that the field of microbial production does not abound in convenient and
recognized techniques.
Much of the thymidine method becomes clear if we take a look at the biochemical background of CH)-TDR uptake and incorporation. The corresponding scheme is given in Fig. 4.12, which explains that microorganisms are
basically "autotrophic" in relation to the DNA nucleoside precursors, because
they possess mechanisms of their de novo synthesis from the intermediates of
tricarbonic acids cycle and amino acids. This de novo way of the thymidine
synthesis (like that of the other nucleosides) is their main source in the microbial cells. It provides normal reproduction of DNA whenever it is needed for
division. This means that actually they have no urgent need for its supply from
external sources, moreover that the incorporation of a large thymidine molecule inside the bacterial cell proceeds with the participation of the permease
enzyme transport mechanism and needs a lot of energy. Only in an emergency
situation, when the bacterial cell has enough energy and resources to proceed
with rapid multiplication, but the de novo synthesis is delayed, may it turn to
the sources of exogenous thymidine, if it is available in surrounding environment. Naturally, in situ it could happen mostly in eutrophic environments,
where thymidine might be present in a concentration over 1 nmol. This
explains why the percentage of bacteria possessing mechanisms necessary
for the uptake of exogenous thymidine is much greater in eutrophic than in
oligotrophic water.
The incorporation of exogenous thymidine (like the uptake of CH)-TDR
label added to the sample) proceeds mainly via the so-called salvation
pathway. The name of this way reflects the emergency character of its use by
the cell. To be able to uptake a large TDR molecule and then to incorporate
it into the DNA, the cell should possess at least two mechanisms very expensive in energy: the permease transport system and the phosphorylase, which
provides the further phosphorylation of uptaken TDR nucleoside molecules
from the ATP (see Fig. 4.12). For bacteria living in low productive waters or
in the mesopelagic layers, where exogenous thymidine is absent, there is no
reason to keep these enzyme systems, while such a reason might be valid in
the euphotic zone of the sea, in lakes, or in bottom sediments. This explains
the wide range of the percentage of bacteria which can incorporate exogenous
CH)-TDR in various aquatic environments, moreover that the method of
radioautography which was employed for this purpose actually misrepresents
the real picture. This happens because, besides the "official" salvation way of
CH)-TDR incorporation, which is complex and energy-expensive, and whose
application is possible only with certain bacterial species of eutrophic habitats,
there is another way, absent from the scheme by Moriarty (1986). This way is
cheap and simple because it is self-sustaining from the energy point of view
and needs only the presence of common permease transport of small mole-
203
above-listed basic assumptions proved to be either not correct or completely
wrong (Karl 1986; Robarts and Zohary 1993). In any case, it is sensible to
salvage something from this method that can be of practical use, in view of the
fact that the field of microbial production does not abound in convenient and
recognized techniques.
Much of the thymidine method becomes clear if we take a look at the biochemical background of CH)-TDR uptake and incorporation. The corresponding scheme is given in Fig. 4.12, which explains that microorganisms are
basically "autotrophic" in relation to the DNA nucleoside precursors, because
they possess mechanisms of their de novo synthesis from the intermediates of
tricarbonic acids cycle and amino acids. This de novo way of the thymidine
synthesis (like that of the other nucleosides) is their main source in the microbial cells. It provides normal reproduction of DNA whenever it is needed for
division. This means that actually they have no urgent need for its supply from
external sources, moreover that the incorporation of a large thymidine molecule inside the bacterial cell proceeds with the participation of the permease
enzyme transport mechanism and needs a lot of energy. Only in an emergency
situation, when the bacterial cell has enough energy and resources to proceed
with rapid multiplication, but the de novo synthesis is delayed, may it turn to
the sources of exogenous thymidine, if it is available in surrounding environment. Naturally, in situ it could happen mostly in eutrophic environments,
where thymidine might be present in a concentration over 1 nmol. This
explains why the percentage of bacteria possessing mechanisms necessary
for the uptake of exogenous thymidine is much greater in eutrophic than in
oligotrophic water.
The incorporation of exogenous thymidine (like the uptake of CH)-TDR
label added to the sample) proceeds mainly via the so-called salvation
pathway. The name of this way reflects the emergency character of its use by
the cell. To be able to uptake a large TDR molecule and then to incorporate
it into the DNA, the cell should possess at least two mechanisms very expensive in energy: the permease transport system and the phosphorylase, which
provides the further phosphorylation of uptaken TDR nucleoside molecules
from the ATP (see Fig. 4.12). For bacteria living in low productive waters or
in the mesopelagic layers, where exogenous thymidine is absent, there is no
reason to keep these enzyme systems, while such a reason might be valid in
the euphotic zone of the sea, in lakes, or in bottom sediments. This explains
the wide range of the percentage of bacteria which can incorporate exogenous
CH)-TDR in various aquatic environments, moreover that the method of
radioautography which was employed for this purpose actually misrepresents
the real picture. This happens because, besides the "official" salvation way of
CH)-TDR incorporation, which is complex and energy-expensive, and whose
application is possible only with certain bacterial species of eutrophic habitats,
there is another way, absent from the scheme by Moriarty (1986). This way is
cheap and simple because it is self-sustaining from the energy point of view
and needs only the presence of common permease transport of small mole-