200
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
enzyme called thymidine kinase, which enables incorporation of CH)TDR
molecules into the DNA. The incorporation rate of CH)-TDR was thought to
reflect microbial growth, which is always combined with the process of DNA
synthesis (Karl 1980,1981). It was also known that de novo DNA synthesis
proceeds only in bacterial cells preparing for cell fission, and that the ability
to incorporate the external thymidine at its nanomolar concentration level is
restricted to bacteria. Other microplankters were assumed to be unable to do
this. These features of CH)-TDR looked very promising for its use for in situ
bacterial production estimations.
The first successful practical attempts in this direction were made
by Brock (1967), who found evidence for the incorporation of exogenous
CH)-TDR into the cells by periphytonic microflora using the microradioautography. Experimentation with its uptake by planktonic bacteria by 1.
Fuprman and F. Azam resulted in the emergence of the thymidine method for
estimating bacterial production in its modern state and protocol. The new
method had many obvious advantages, delineated by its just proponents,
such as its high sensitivity and selectivity, a minor disturbance of bacterioplankton during the experiments, which are short-term and accomplished
without the addition of easily utilizable organic substrates into the natural
samples by bacteria.
The assumed direct permease mechanism of transport intact TDR
molecules inside the cells and their subsequent incorporation into the
DNA gave hopes at that time of finding a universal translation factor between
the dpm radioactivity of incorporated CH) TDR and the production or
fission rate of bacteria in their natural populations. Being well aware of the
advantages afforded by the new method, its proponents had made certain
assumptions, concerning features of CH)-TDR uptake and its incorporation into DNA by natural bacterioplankton populations, which made this
method reliable and applicable for a variety of aquatic environments. At that
time these assumptions sounded quite valid. The most important among them
were:
1. A majority of heterotrophic bacteria are capable of incorporating external
TDR in their ambient populations to replenish demand in DNA presecutors during the process of their division and growth.
2. Minor concentrations of free TDR, which are present in natural water
samples «1 nmol), could cause only a minor isotope dilution effect upon
its pool, created by the addition of CH)-TDR in them (5-30nmol).
3. Its concentrations used in the experiments up to 10-30nmol are able to
block or reduce the isotopic dilution effect resulting from the de novo
thymidine synthesis (see Fig. 4.12). Thus the dilution of CH)-TDR from the
internal and external sources is negligible.
4. Thymidine constitutes a constant fraction in the bacterial DNA of about
25%, and the DNA itself constitutes a constant fraction in bacterial
biomass.
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
enzyme called thymidine kinase, which enables incorporation of CH)TDR
molecules into the DNA. The incorporation rate of CH)-TDR was thought to
reflect microbial growth, which is always combined with the process of DNA
synthesis (Karl 1980,1981). It was also known that de novo DNA synthesis
proceeds only in bacterial cells preparing for cell fission, and that the ability
to incorporate the external thymidine at its nanomolar concentration level is
restricted to bacteria. Other microplankters were assumed to be unable to do
this. These features of CH)-TDR looked very promising for its use for in situ
bacterial production estimations.
The first successful practical attempts in this direction were made
by Brock (1967), who found evidence for the incorporation of exogenous
CH)-TDR into the cells by periphytonic microflora using the microradioautography. Experimentation with its uptake by planktonic bacteria by 1.
Fuprman and F. Azam resulted in the emergence of the thymidine method for
estimating bacterial production in its modern state and protocol. The new
method had many obvious advantages, delineated by its just proponents,
such as its high sensitivity and selectivity, a minor disturbance of bacterioplankton during the experiments, which are short-term and accomplished
without the addition of easily utilizable organic substrates into the natural
samples by bacteria.
The assumed direct permease mechanism of transport intact TDR
molecules inside the cells and their subsequent incorporation into the
DNA gave hopes at that time of finding a universal translation factor between
the dpm radioactivity of incorporated CH) TDR and the production or
fission rate of bacteria in their natural populations. Being well aware of the
advantages afforded by the new method, its proponents had made certain
assumptions, concerning features of CH)-TDR uptake and its incorporation into DNA by natural bacterioplankton populations, which made this
method reliable and applicable for a variety of aquatic environments. At that
time these assumptions sounded quite valid. The most important among them
were:
1. A majority of heterotrophic bacteria are capable of incorporating external
TDR in their ambient populations to replenish demand in DNA presecutors during the process of their division and growth.
2. Minor concentrations of free TDR, which are present in natural water
samples «1 nmol), could cause only a minor isotope dilution effect upon
its pool, created by the addition of CH)-TDR in them (5-30nmol).
3. Its concentrations used in the experiments up to 10-30nmol are able to
block or reduce the isotopic dilution effect resulting from the de novo
thymidine synthesis (see Fig. 4.12). Thus the dilution of CH)-TDR from the
internal and external sources is negligible.
4. Thymidine constitutes a constant fraction in the bacterial DNA of about
25%, and the DNA itself constitutes a constant fraction in bacterial
biomass.