Determination of Microbial Production
205
radioactivity, with range its share ranging between 0 and 90% (Riemann et al.
1982,1987; Scavia et al. 1986; Servais et al. 1987; Cho and Azam 1988; Smith
and Riemann 1988; Hollibaugh 1988; Carman et al. 1988; Brittain and Karl
1990). This share can be very variable, depending on many factors starting
from the practical protocol of the experiment and ending with various environmental tactors. It makes the estimatiun uf the rate of DNA synthesis with
this method quite unreasonable. Similarly, it makes the attempts to derive any
stable conversion factor translatable to microbial production useless, moreover, the principal assumption that only growing bacterial cells incorporate
exogenous thymidine also turned out to be incorrect (Bloem et al.1988; Davis
1989).
The remaining, and most often the larger part of the cold TCA-insoluble
macromolecular fraction is represented by RNA and proteins. Also a
significant part of uptaken CH)-label from TDR by the microbial cells was discovered in the lipids (Carman et al. 1988; Robarts and Wicks 1989); but via the
restricted salvation pathway the thymidine nucleoside molecules theoretically
are permitted to be incorporated directly and only into DNA. Can it happen
that the labeled become different substances like proteins or RNA, and even
labeled more than the DNA itself? Now we are facing the phenomenon of
unspecific labeling, which is one of "black cats" of the thymidine method. The
phenomenon itself appears as follows. The CH)-TDR added into the natural
samples in concentrations of 5-30nm within 10-30min of exposure
significantly labels bacterioplankton cells, not only with DNA but also with
other macromolcules, like proteins, RNA, and lipids. The level of labeling as
the percentage of total radioactivity discovered in the macromolecular traction most often varies, between 2 and 90% in DNA, between 20 and 98% in
RNA and between 20 and 70% in proteins. The label of 3H may be transferred
from the CH)-methyl thymidine to those compounds only during the traditional cellular pathways of their de novo synthesis. How could it then proceed
in practical CH)-TDR experiments with bacterioplankton? The first explanation was, that it could happen only during long exposures (over 1520min) when the microbial cells using the salvation pathway accumulate an
excess of thymidine precursors which cannot be used immediately for DNA
synthesis. This excess of 3H-thymidine nucleotide is disintegrated inside the
cell with the participation of corresponding enzymes down to the carbonic
acids and amino acids bearing the label, with the liberation of some amount
of 3H-methyl groups. The residues of 3H-TDR enter the cellular metabolism,
which results in the appearance of 3H label in RNA, proteins, and lipids. Later
evidence was, however, obtained, that this way is not the only one, and, most
probably, not the main one for nonspecific labeling (Carman et al. 1988; Davis
1989; Brittain and Karl 1990).
The other way is the catabolic pathway, when the permanently hungry
oligocarbophylic bacteria, composing the majority of microbial populations in
water columns, simply use the CH)-TDR as food when it is added to the
samples in significant amounts of to 5 to 20 nmol during thymidine productiv-
205
radioactivity, with range its share ranging between 0 and 90% (Riemann et al.
1982,1987; Scavia et al. 1986; Servais et al. 1987; Cho and Azam 1988; Smith
and Riemann 1988; Hollibaugh 1988; Carman et al. 1988; Brittain and Karl
1990). This share can be very variable, depending on many factors starting
from the practical protocol of the experiment and ending with various environmental tactors. It makes the estimatiun uf the rate of DNA synthesis with
this method quite unreasonable. Similarly, it makes the attempts to derive any
stable conversion factor translatable to microbial production useless, moreover, the principal assumption that only growing bacterial cells incorporate
exogenous thymidine also turned out to be incorrect (Bloem et al.1988; Davis
1989).
The remaining, and most often the larger part of the cold TCA-insoluble
macromolecular fraction is represented by RNA and proteins. Also a
significant part of uptaken CH)-label from TDR by the microbial cells was discovered in the lipids (Carman et al. 1988; Robarts and Wicks 1989); but via the
restricted salvation pathway the thymidine nucleoside molecules theoretically
are permitted to be incorporated directly and only into DNA. Can it happen
that the labeled become different substances like proteins or RNA, and even
labeled more than the DNA itself? Now we are facing the phenomenon of
unspecific labeling, which is one of "black cats" of the thymidine method. The
phenomenon itself appears as follows. The CH)-TDR added into the natural
samples in concentrations of 5-30nm within 10-30min of exposure
significantly labels bacterioplankton cells, not only with DNA but also with
other macromolcules, like proteins, RNA, and lipids. The level of labeling as
the percentage of total radioactivity discovered in the macromolecular traction most often varies, between 2 and 90% in DNA, between 20 and 98% in
RNA and between 20 and 70% in proteins. The label of 3H may be transferred
from the CH)-methyl thymidine to those compounds only during the traditional cellular pathways of their de novo synthesis. How could it then proceed
in practical CH)-TDR experiments with bacterioplankton? The first explanation was, that it could happen only during long exposures (over 1520min) when the microbial cells using the salvation pathway accumulate an
excess of thymidine precursors which cannot be used immediately for DNA
synthesis. This excess of 3H-thymidine nucleotide is disintegrated inside the
cell with the participation of corresponding enzymes down to the carbonic
acids and amino acids bearing the label, with the liberation of some amount
of 3H-methyl groups. The residues of 3H-TDR enter the cellular metabolism,
which results in the appearance of 3H label in RNA, proteins, and lipids. Later
evidence was, however, obtained, that this way is not the only one, and, most
probably, not the main one for nonspecific labeling (Carman et al. 1988; Davis
1989; Brittain and Karl 1990).
The other way is the catabolic pathway, when the permanently hungry
oligocarbophylic bacteria, composing the majority of microbial populations in
water columns, simply use the CH)-TDR as food when it is added to the
samples in significant amounts of to 5 to 20 nmol during thymidine productiv-