DNA and Protein Synthesis - Indicators of microbial activity in soil represent measurements at the ecosystem level (e.g. processes regulating decomposition of organic residues and nutrient cycling, especially nitrogen, sulphur, and phosphorus). Measurements at the community level include bacterial DNA and protein synthesis. Frequency of bacteriophages is a measurement at the population level.
Bacterial DNA synthesis
Synthesis of DNA is a prerequisite for bacterial cell division and, as such, an indicator of bacterial growth. DNA is unique in the way that it only participates in cell division. DNA synthesis can be determined by incorporation of 3H- or 14C-thymidine into bacterial DNA as thymidine is a unique nucleoside, which only participates in DNA synthesis.
The method is used routinely in the Dutch Soil Monitoring Programme and has been shown to discriminate between different soil types and land uses, e.g. grassland on clay and horticultural farm on sand (Schouten et al. 1999). Bacterial growth rate (number of cells formed per unit time) is calculated by use of a conversion factor (Michel et al. 1993). This conversion factor is based on many assumptions, including estimates of the number of cells present and the amount of radiolabelled thymidine incorporated in relation to GC content of the total DNA content of cells.
Bacterial protein synthesis
Bacterial protein synthesis is directly correlated to bacterial activity and can be determined by incorporation of 3H or 14C leucine, as this amino acid is incorporated into proteins only. The method for leucine incorporation is the same as for thymidine incorporation (see above) and the incorporation of both precursors can be carried out in a single assay if different radiolabels are used (Bloem et al. 2002). Incorporation of 14C leucine is routinely measured in the Dutch Soil Monitoring Programme in combination with 3H-thymidine incorporation (Bloem et al. 2002) and has been shown to possess discriminative power (Schouten et al. 1999).
The advantages and drawbacks of the method are the same as for radiolabelled thymidine incorporation, although balanced growth is not a prerequisite. Furthermore, most bacteria take up leucine, although the incorporation efficiency may differ between soils (. Measurements of protein synthesis are supposed to be more accurate than that of DNA synthesis, because of a relatively higher protein content in cells.
RNA measurements
The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA), play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis. Quantification of activity by either method is still problematic and comprehensive method development is needed before implementation into a monitoring programmed.
Bacteriophages
A bacteriophage is a virus, which infects and multiplies in a specific host bacterium. Bacteriophages are abundant in the soil environment and have been isolated for nearly every known species of soil bacteria. Most phages isolated from soil are temperate phages, e.g. phages that can lie dormant in bacterial cells after infection. The multiplication of bacteriophages strictly depends on the activity of the host bacteria. As such, monitoring of the frequency and host specificity of free bacteriophages in soil is an indicator of the activity of specific soil bacteria. This is in contrast to the other microbial activity indicators, which measure the activity of whole microbial communities.
Determination of free bacteriophages in soil can be carried out by a standard method of extraction followed by a plaque-assay (e.g. (Hu 1998)) with specific host bacteria, e.g. Pseudomonas (Cambell et al.1995), Bacillus (Pantasticocaldas et al. 1992), Rhizobium (Radeva et al. 2001). A high number of plaques are presumed to indicate a recent high activity of similar host bacteria in the test soil assuming a direct correlation between the number of bacteriophages and bacterial activity. Such a correlation has indeed been shown for Azospirillum brasilense (microcosm study; (Germida 1986)) and Serratia liquefaciens (field study; (Ashelford et al. 2000)), but has to be confirmed for other bacterial groups.
The selection of host bacteria should be representative for the soil type to be investigated. Furthermore, the bacteriophage sensitivity to the host bacteria should be known. The frequency and persistence of the bacteriophages in different soil types should be estimated a priori in order to standardize the method. Generally, temperate bacteriophages survive for long periods of time within the host bacteria. Without host bacteria, the survival of bacteriophages depends on abiotic parameters, e.g. clay content, soil moisture, temperature and pH.
Bacterial DNA synthesis
Synthesis of DNA is a prerequisite for bacterial cell division and, as such, an indicator of bacterial growth. DNA is unique in the way that it only participates in cell division. DNA synthesis can be determined by incorporation of 3H- or 14C-thymidine into bacterial DNA as thymidine is a unique nucleoside, which only participates in DNA synthesis.
The method is used routinely in the Dutch Soil Monitoring Programme and has been shown to discriminate between different soil types and land uses, e.g. grassland on clay and horticultural farm on sand (Schouten et al. 1999). Bacterial growth rate (number of cells formed per unit time) is calculated by use of a conversion factor (Michel et al. 1993). This conversion factor is based on many assumptions, including estimates of the number of cells present and the amount of radiolabelled thymidine incorporated in relation to GC content of the total DNA content of cells.
Bacterial protein synthesis
Bacterial protein synthesis is directly correlated to bacterial activity and can be determined by incorporation of 3H or 14C leucine, as this amino acid is incorporated into proteins only. The method for leucine incorporation is the same as for thymidine incorporation (see above) and the incorporation of both precursors can be carried out in a single assay if different radiolabels are used (Bloem et al. 2002). Incorporation of 14C leucine is routinely measured in the Dutch Soil Monitoring Programme in combination with 3H-thymidine incorporation (Bloem et al. 2002) and has been shown to possess discriminative power (Schouten et al. 1999).
The advantages and drawbacks of the method are the same as for radiolabelled thymidine incorporation, although balanced growth is not a prerequisite. Furthermore, most bacteria take up leucine, although the incorporation efficiency may differ between soils (. Measurements of protein synthesis are supposed to be more accurate than that of DNA synthesis, because of a relatively higher protein content in cells.
RNA measurements
The RNA molecules, ribosomal RNA (rRNA) and messenger RNA (mRNA), play key roles in the protein synthesis. The amount of RNA in individual cells or in a community may, therefore, be taken as an indicator of protein synthesis and, thus, microbial activity. The number of active cells can be detected by fluorescent in situ hybridization (FISH). By this method, individual cells carrying high concentrations of rRNA, situated on ribosomes, are quantified by fluorescence microscopy. The amount of rRNA in a community can also be detected by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), where rRNA extracted from soil is detected by creating a DNA copy and separating by gel electrophoresis. Quantification of activity by either method is still problematic and comprehensive method development is needed before implementation into a monitoring programmed.
Bacteriophages
A bacteriophage is a virus, which infects and multiplies in a specific host bacterium. Bacteriophages are abundant in the soil environment and have been isolated for nearly every known species of soil bacteria. Most phages isolated from soil are temperate phages, e.g. phages that can lie dormant in bacterial cells after infection. The multiplication of bacteriophages strictly depends on the activity of the host bacteria. As such, monitoring of the frequency and host specificity of free bacteriophages in soil is an indicator of the activity of specific soil bacteria. This is in contrast to the other microbial activity indicators, which measure the activity of whole microbial communities.
Determination of free bacteriophages in soil can be carried out by a standard method of extraction followed by a plaque-assay (e.g. (Hu 1998)) with specific host bacteria, e.g. Pseudomonas (Cambell et al.1995), Bacillus (Pantasticocaldas et al. 1992), Rhizobium (Radeva et al. 2001). A high number of plaques are presumed to indicate a recent high activity of similar host bacteria in the test soil assuming a direct correlation between the number of bacteriophages and bacterial activity. Such a correlation has indeed been shown for Azospirillum brasilense (microcosm study; (Germida 1986)) and Serratia liquefaciens (field study; (Ashelford et al. 2000)), but has to be confirmed for other bacterial groups.
The selection of host bacteria should be representative for the soil type to be investigated. Furthermore, the bacteriophage sensitivity to the host bacteria should be known. The frequency and persistence of the bacteriophages in different soil types should be estimated a priori in order to standardize the method. Generally, temperate bacteriophages survive for long periods of time within the host bacteria. Without host bacteria, the survival of bacteriophages depends on abiotic parameters, e.g. clay content, soil moisture, temperature and pH.
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