204
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
cules for ordinary cellular biosynthesis (Carman et al. 1988; Brittain and Karl
1990). Here we are talking of the catabolism pathway (Fig. 4.12). The CH)TDR molecules are accessible as food for the "paupers" - the oligocarbophylic
aquatic bacteria - which, being good survivors, are able to break down the
large complex nucleoside molecules (Davis 1989). Being added to the water
samples in a significant concentration of 30-60 nnlOl to obtain good labeling
for radioautography, CH)-TDR is absorbed by oligocarbophylic bacteria and
catabolized by them as an ordinary organic substrate down to small molecules
like glutamate or oxaloacetate, which carry the 3H-Iabel. The latter are, in part
oxidized, but in part they also are transported inside the cell and participate
in constructive cellular metabolism, entering the de novo synthesis of DNA
precursors, such as thymidine. Because of the high specific radioactivity of the
initial CH)-TDR added, they can in this way acquire enough label to be
recorded as actively incorporating the exogenous TDR and logistically possessing necessary for that transport system and thymidine kinase; but employing this approach, which evidently may seriously overestimate the percentage
of bacterioplankton cells able to incorporate exogenous thymidine via the
rescue way, this percentage appeared to be only 10 to 30% even in a eutrophic
lake (Douglas et al. 1987; Pedros Alio and Newell 1989). In other habitats it
could drop down to 1 %.
Experiments with cultures of marine bacteria showed that a majority of
the most common Pseudomonas species among them do not possess the
thymidine kinase and because of this are incapable of direct TOR uptake
(Carlson et al. 1985; Saito 1985; Jeffrey and Paul 1988; Davis 1989), like many
species among the actinomicetes, yeasts, and mycobacteria, which are common
components of bacterioplankton communities. The uptake mechanisms are
absent also in chemoautotrophic bacteria, which are important producers
of microbial biomass, especially the group of methylotrophs (Mc Donaugh
et al. 1986; Johnstone and Jones 1989), as also is in the majority of anaerobic
bacteria (Winding 1992). Thus one of the basic assumptions mentioned above
about the predomination in natural habitats of bacteria incorporating exogenous thymidine for DNA synthesis was not proved correct. Variations in
even small percentages in natural habitats result in variations of the conversion factor.
Another important parameter needed for the stability of the conversion
factor is the constant DNA content in the bacterial cells in relation to their
biomass or protein; but in real natural bacterial assemblages it varies between
two and three times (Cho and Azam 1988). The same variations occur within
a rather large range (30 to 70%), the share of the thymidine base in bacterial
DNA (Cho and Azam 1988), which again results in variability of the conversion factor. An even greater confusion in the interpretation of labeling
natural bacterial assemblages with eH)-thymidine was, however, created by
the discovery that in the cold TCA-resistant macromolecular fraction, separated within the protocol of thymidine method for measuring the radioactivity of DNA in it, the CH)-TDR most often represents a minor part of its
Use of Radioisotopic Methodology in Aquatic Microbial Ecology
cules for ordinary cellular biosynthesis (Carman et al. 1988; Brittain and Karl
1990). Here we are talking of the catabolism pathway (Fig. 4.12). The CH)TDR molecules are accessible as food for the "paupers" - the oligocarbophylic
aquatic bacteria - which, being good survivors, are able to break down the
large complex nucleoside molecules (Davis 1989). Being added to the water
samples in a significant concentration of 30-60 nnlOl to obtain good labeling
for radioautography, CH)-TDR is absorbed by oligocarbophylic bacteria and
catabolized by them as an ordinary organic substrate down to small molecules
like glutamate or oxaloacetate, which carry the 3H-Iabel. The latter are, in part
oxidized, but in part they also are transported inside the cell and participate
in constructive cellular metabolism, entering the de novo synthesis of DNA
precursors, such as thymidine. Because of the high specific radioactivity of the
initial CH)-TDR added, they can in this way acquire enough label to be
recorded as actively incorporating the exogenous TDR and logistically possessing necessary for that transport system and thymidine kinase; but employing this approach, which evidently may seriously overestimate the percentage
of bacterioplankton cells able to incorporate exogenous thymidine via the
rescue way, this percentage appeared to be only 10 to 30% even in a eutrophic
lake (Douglas et al. 1987; Pedros Alio and Newell 1989). In other habitats it
could drop down to 1 %.
Experiments with cultures of marine bacteria showed that a majority of
the most common Pseudomonas species among them do not possess the
thymidine kinase and because of this are incapable of direct TOR uptake
(Carlson et al. 1985; Saito 1985; Jeffrey and Paul 1988; Davis 1989), like many
species among the actinomicetes, yeasts, and mycobacteria, which are common
components of bacterioplankton communities. The uptake mechanisms are
absent also in chemoautotrophic bacteria, which are important producers
of microbial biomass, especially the group of methylotrophs (Mc Donaugh
et al. 1986; Johnstone and Jones 1989), as also is in the majority of anaerobic
bacteria (Winding 1992). Thus one of the basic assumptions mentioned above
about the predomination in natural habitats of bacteria incorporating exogenous thymidine for DNA synthesis was not proved correct. Variations in
even small percentages in natural habitats result in variations of the conversion factor.
Another important parameter needed for the stability of the conversion
factor is the constant DNA content in the bacterial cells in relation to their
biomass or protein; but in real natural bacterial assemblages it varies between
two and three times (Cho and Azam 1988). The same variations occur within
a rather large range (30 to 70%), the share of the thymidine base in bacterial
DNA (Cho and Azam 1988), which again results in variability of the conversion factor. An even greater confusion in the interpretation of labeling
natural bacterial assemblages with eH)-thymidine was, however, created by
the discovery that in the cold TCA-resistant macromolecular fraction, separated within the protocol of thymidine method for measuring the radioactivity of DNA in it, the CH)-TDR most often represents a minor part of its