Future Prospects

Nucleic acid based methods for assessment of the environmental fate and effects of bacteria are under continuous development. In the future, growth and wider availability of nucleic acid sequence databases, further improvement and optimization of molecular techniques as well as application of microarray chips will facilitate monitoring studies.

DNA databases are of great help in probe and primer design. Since data are accumulating very fast in nucleic acid sequence databanks, special databases of probes and PCR primers are of great value. There are probe databases accessible via the Internet. Alm et al have established an oligonucleotide probe database.63 Their probes and primers, mainly based on rRNA sequences, are widely used in microbial ecology and environmental microbiology. The authors also provide advice on probe and PCR primer design and characterization. A more complete database for ribosomal sequences and for data analysis tools is maintained at the Center for Microbial Ecology at Michigan State University.64 Another database is available at the Technical University of Munich (http://www.mikro.biologie.tu-muenchen.de).

One of the most p-omising techniques is the direct detection and quantification of gene transcripts (mRNA) by reverse transcription-PCR, which allows assessment of micro-

Fig. 4.5. REP PCR fingerprint patterns of R. galegae DNA obtained by sonicating root nodules. Lanes: S, lambda BstEII; 1, genomic DNA from reference strain R. galegae HAMBI 540; 2, field nodule (R. galegae HAMBI 540); 3-8, nodules from G. orientalis inoculated with different R. galegae strains; 9, negative control without template DNA. Reprinted with permission from Nick G, Lindström K. System Appl Microbiol 1994; 17:265-273.

Fig. 4.5. REP PCR fingerprint patterns of R. galegae DNA obtained by sonicating root nodules. Lanes: S, lambda BstEII; 1, genomic DNA from reference strain R. galegae HAMBI 540; 2, field nodule (R. galegae HAMBI 540); 3-8, nodules from G. orientalis inoculated with different R. galegae strains; 9, negative control without template DNA. Reprinted with permission from Nick G, Lindström K. System Appl Microbiol 1994; 17:265-273.

bial activities in the field (For a review see ref 65). The RT-PCR technique consists of synthesis of DNA from RNA by reverse transcription, and amplification of a specific DNA fragment by PCR. The method is very sensitive, and by using appropriate controls the detection can be made quantitative. The first environmental applications included detection of food-borne pathogens,66 and microorganisms used in bioremediation of contaminated soils.65

For the selection of a suitable target gene for RT-PCR several characteristics should be considered: the target gene should be abundantly expressed throughout the growth cycle of the organism tested, and its expression shoild not be regulated at the transcriptional level. Since bacterial mRNAs have a very short half-life, these methods discriminate living (i.e., metabolically active) cells from dead or domant ones, although DNA from the latter ones can still be detected by DNA-targeted Southern hybridization or amplified by conventional PCR. (Detection of active contra dormant cells is discussed in more detail in Chapter 1.) Some technical difficultes arise from the need for rapid isolation of undegraded mRNA. However, in situ techniques are now under development for the amplification of gene transcripts inside microbial cells.18

In addition, the fingerprints achieved by the PCR-based applications mentioned in section 4.2.1 can be used for identification of microorganisms in pure cultures or relatively simple microbial communities. In highly diverse microbial systems such as soil, the banding patterns are too complex to be assigned to specific organisms but a comparison of micro-bial communities (diversity as well as quantitative aspects)67 is possible. Particularly the TGGE technique seems to be appropriate for monitoring effects of environmental disturbances such as pollution or release of new organisms.68

Hybridization of RNA and DNA on microarrayed 'chips' (on glass or silica surfaces) is another technique that will probably be broadly applied in the near future. Cheng et al prepared nucleic acids of E. coli and other bacteria from human blood and hybridized them with appropriate probes on microchips.69 Since the hybridization of RNA on microarrayed chips allows monitoring of gene expression, this technique may prove to be useful in studying the dynamics of cell populations in environmental samples. Thus, microarrays have great potential in medical diagnostics, food monitoring, water testing, and other fields.

New approaches based on specific molecular recognition mechanisms are also under development, such as nucleic acid probes detecting proteins and other molecules,70 or synthetic DNA mimics that behave like DNA oligonucleotides but bind to nucleic acids with higher specificity and affinity.71 Drolet et al have developed an ELISA-like assay using an oligonucleotide probe which binds to a protein target.70 A method called SELEX (systematic evolution of ligands by exponential enrichment)72 allowed rapid selection of oligonucleotides that preferentially bind to the target molecule from a population of random sequences. High affinity oligonucleotide ligands could be developed in this way for virtually any target molecule and used, for example, as therapeutic or diagnostic agents.73 Nucleic acid ligands would have several advantages over antibodies.70 For example, they can be easily synthesized and thus accurately replicated with the same binding properties, while antibodies may differ when generated in different laboratories and are also subject to animal-to-animal variation. Secondly, animals are not required for the synthesis of oligonucleotide ligands. Also, the ligands can be generated even for toxic or nonimmunogenic targets. Their small size can also be an advantage for some applications. In the future, further DNA ligands will be developed for diagnostic purposes and may be used for more general approaches. If successful, the method could challenge, for example, in situ hybridization and immune detection methods.

An interesting new approach is based on the use of a synthetic molecule peptide nucleic acid (PNA). This mdecule has recently been proposed to expand the applications of oligonucleotides.71 PNAs are DNA analogs with a polyamide backbone substituted with purine and pyrimidine base sidechains. The specificity of PNA in binding to complementary nucleic acids is higher than that of DNA strands, and the resulting PNA/DNA and PNA/RNA duplexes have high stability compared to DNA/DNA and DNA/RNA duplexes. Similarly to DNA, PNA can be labeled by biotin, fluorescein or reporter enzymes. Therefore, PNA is a good candidate for hybridization studies and may, one day, share the burgeoning field of DNA-based diagnostics with traditional nucleic acid probes.

Along with the above described sophisticated genetic approaches, phenotypic description of microbial communities is equally important. Metabolic fingerprints of individual organisms or microbial communities can be determined by the BIOLOG' system, which colorimetrically detects the utilization of various carbon substrates. Phospholipid fatty acid (PLFA) profiles are also increasingly being used to characterize the structure of microbial communities.74,75

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