- Email: [email protected]
Nucleic acids in the environment
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Nucleic acids in the environment Jack T Trevors The past year has witnessed several excellent advances in basic and applied research on nucleic acids in the environment. Improved methods for extracting nucleic acids from environmental samples have been published, as well as information on the use of reporter genes in bacteria, natural genetic transformation in soil and DNA adsorption to soil. These advances will have a significant impact on both future research and the way in which we view nucleic acids in the environment.
Address Laboratory of Microbial Technology, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:331-336 © Current Biology Ltd ISSN 0958-1669 Abbreviations GEM geneticallyengineered microorganism GFP green fluorescent protein
Introduction
Nucleic acids (i.e. DNA and RNA) are virtually ubiquitous in the biosphere. If you look past the soil, water, sediment, vegetation and animals on the Earth, and imagine the amount of DNA necessary to encode for the multitude of living organisms, an appreciation of the amount of D N A and RNA in the environment can be obtained. For example, Selenska and Klingmuller [1] reported that - 3 0 0 n g DNA and - 1 0 0 n g RNA can be extracted from 10g soil. This value does not include DNA/RNA in the rhizosphere, other plant-derived DNA/RNA or DNA/RNA from organisms above the soil surface. T h e Earth is virtually covered with nucleic acids. Not only is the amount of DNA on the Earth overwhelming, but its diversity and ability to undergo genetic recombination and mutation while retaining a degree of constancy illustrate both the complexity of life on this planet and the evolutionary path(s) that have brought life on Earth to its present state. Researchers and students of environmental microbiology (and other scientific disciplines) are poised at the beginning of an era in which the use of molecular biology in their field is still in its infancy. For example, the use of PCR in environmental microbiology was reviewed in 1994 by Bej and Mahbubani [2]. T h e high specificity, sensitivity and reproducible consistency of PCR detection of specific DNA sequences in complex environmental samples has contributed significantly to the advancement of knowledge in environmental microbiology and many other areas of research (e.g. detection of food-borne microbial pathogens, clinical diagnostics, forensic science, genome mapping, plant taxonomy and evolution, detec-
tion of mutations, mutagenesis by PCR, generation of DNA probes by PCR, and the cloning of PCR products) [3]. T h e past year has seen numerous improvements to, and applications of, PCR in environmental microbiology, especially with soil, sediment, water and plant material. A recently published text [4"] includes information on the theory and protocols for the extraction of DNA and RNA from environmental samples and covers developments from the past 15 years. T h e majority of the research and methods has, however, originated in the past 8 years. This review examines recent advances in nucleic acids in the environment from an environmental microbiology/biotechnology perspective. To date, most of the research has focused on DNA, with less emphasis on RNA extraction and purification. Yet mRNA can now be extracted from environmental samples such as soil [5] and sediment [6"]. Although these methods were introduced before the past year, they are significant to the area of nucleic acids in the environment and, therefore, are cited here. Other research discussed in this review includes reporter gene technology, genetic transformation in the environment and DNA adsorption to soil (see Table 1). Direct DNA extraction
and PCR amplification
Direct soil DNA extraction techniques are now an important part of microbial ecology research. These methods are useful in the investigation of indigenous bacteria or bacteria introduced into the environment. Because viable microbial cells can be only partially recovered from complex environmental samples by traditional viable plating methods, cell extraction from soil and sediment samples recovers only a fraction of the cells present. Also, a large fraction, often 90-99%, of microbial cells present in environmental samples are not culturable on microbiological media and, therefore, escape detection when cultivation-based methods are used. Because a large percentage of microbial cells in environmental samples may be viable but non-culturable, their detection via molecular techniques is often necessary. Only in recent years has microbial DNA in the environment been studied using the powerful technology of PCR. Complex environmental samples, such as soil, sediment and water, from which D N A (or RNA) is extracted contain a multitude of substances (e.g. clays, metals, toxic chemicals, nucleases and humic material) that can inhibit the PCR reaction. For this reason, environmental samples must be highly purified to be used as templates in PCR analysis. It is now possible to obtain highly purified DNA from environmental samples in short periods of time (several hours) that can be used for PCR analysis. T h e
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Table 1
Figure 1
Some recent research on nucleic acids In the environmenL
Research Direct extraction and PCR Extraction, purification and analysis of DNA from soil bacteria Extraction and amplification of DNA from rhizophere and rhizoplane Extraction of DNA from phyllosphere Specific DNA sequences for detection of soil bacteria PCR amplification of DNA from root nodules Extraction of DNA/RNA from aquatic environments PCR amplification of DNA from aquatic environments Extraction and amplification of 16S rRNA genes from deep marine sediments and seawater PCR for detection of antibiotic resistance genes in environmental samples DNA analysis to determine diversity of microbial communities Detection of DNA sequences in environmental samples by PCR Isolation, purification of bacterial DNA from soil Reporter genes Survival of lux-lac marked P. aeruginosa UG2 in soil monitored by PCR Survival of carrageenan-encapsulated and unencapsulated P. aeruginosa UG2Lr in forest soil monitored by PCR and viable plating Molecular marker systems for detecting GEMs in the environment PCR detection of luxAB-marked Alcaligenes eutrophus H850 polychlorinated biphenyl contaminated soil and sediment Natural transformation Effect of divalent cations on natural transformation of P. stutzeri in marine sediment DNA in soil: adsorption, genetic transformation and molecular evolution Bacterial gene transfer by natural genetic transformation in the environment DNA adsorption to soil Effects of DNA polymer length on its adsorption to soils Molecular evolution In bacteria Information on molecular evolution of bacteria, including microbiology, molecular biology, metal-microbe interactions and restrictionmodification systems
References [22] [8] [23] [24] [25] [26] [27] [28] [29] [9"] [30] [31] [11 ] [13] [10",32] [12]
[8] [23]
Generalized procedure for direct DNA extraction from soil or sediment. One advantage of this procedure is that only a small sample (0.5-1.0g) is required and the DNA is extracted directly from the sample without first extracting the microbial cells from the sample. It is necessary to achieve good lysis of the microbial cells in samples to ensure the DNA under study is released and recovered. This is especially important if the number of cells containing the DNA of interest is low. Once the cells are lysed, the extract can be subjected to additional purification procedures using a spun column and glass-milk clean-up procedures. The recovered DNA can then be quantified and used for PCR analysis or other laboratory manipulations.
[24]
[181
[33]
ability to obtain amplifiable D N A from D N A extracts directly is significant given that the ability to recovery bacterial cells from soil can range from about 7% to 18% [7] and often is lower. Figure 1 summarizes the main steps in the direct extraction of DNA from soil or sediment. Most methods add phosphate or Tris buffers to soil, followed by rapid mixing (several hundred to thousands of rpm) for -5-30rain at room temperature to initially produce a slurry in which subsequent lysis of cells followed by
extraction and purification of DNA can be achieved. Some methods use repeated freezing (--80"C) and thawing (room temperature or higher) cycles to achieve lysis of the microbial cells. T h e extract obtained is generally sufficiently pure to use as a template in PCR reactions. T h e additional purification using glass milk (Geneclean II, Bio 101 Inc, La Jolla, California, USA) reduces the DNA yield, but provides highly purified DNA that can be used as a D N A template. One recent approach is the use of magnetic beads (Dynabeads M-280 streptavidin, Dynal Inc, Lake Success, New York, USA) linked to a single-stranded specific DNA probe for selective recovery of complementary target sequences from samples, which are subsequently amplified for detection and/or isolation. Nucleic acids obtained from complex environmental samples should ideally be representative of the biota present. With these samples, it is also necessary to assess both the quality and quantity of the nucleic acids obtained. T h e quantity of extracted and purified DNA can be assessed on agarose gels and compared with DNA size ladders.
Nucleic acids in the environment Trevors 333
DNA quantification based on spectrophotometric analysis at 260nm does not work well for soil-derived D N A because of the presence of strongly absorbing compounds in soil. Alternative methods include fiuorometric determination of DNA using Hoechst dye 33258 (bisbenzimide: 2-[2-(4-hydroxyphenyl)-6-benzimidazoly)]6-(1-methyl-4-piperazyl) benzmidazole-3HCl pentahydrate [Calbiochem Diagnostics, La Jolla, California, USA]) and ethidium bromide (2,7-diamino-10-ethyly9-phenyl-phenanthridinium bromide [8]. Fluorescence measurements using Hoechst 33258 can be measured using a Hoefer 100 Mini-fluorometer (Hoefer Scientific Instruments, San Francisco, California, USA) calibrated with purified EscheHchia coli DNA (Sigma Chemical Company, St Louis, USA). DNA concentrations in the nanogram range can be quantified. A 2 nl sample is adequate for the determination; therefore, most of the sample is available for other analyses. DNA concentrations are expressed as nanogram per gram of dry soil/sediment/root or nanogram per milliliter of water. Another fluorometric method for quantification of D N A employs ethidium bromide as the fluorescent dye and a Tyler Model SSF-600 solid-state fluorometer (Tyler Research, Edmonton, Alberta, Canada) to measure fluorescence [81. Ethidium bromide absorbs light at both 280 nm and 520 nm, with excitation of either wavelength giving rise to emission at 600nm. T h e SSF-600 excites at 520nm and measures the emission at 600nm. A third method for assessing the DNA content of soil is based on scanning (using a Pharmacia LKB UltroScan XL Scanner) of pictures of ethidium bromide stained DNA bands in agarose gels and their comparison with pure D N A standards. This method has the advantage of electrophoretic removal of interference by humic material (electrophoresed DNA is highly purified). It is also possible to use the ability to successfully digest the DNA with restriction enzymes or the ability to amplify a target sequence by PCR, as measures of DNA purity. With respect to thermal-cycling equipment, a fast capillary thermal sequencer (FTS-IS; Corbett Research, New South Wales, Australia) can achieve 30 cycles in 30 min and does not require an oil overlay. This permits direct loading of reagents and samples in a capillary tube, which is sealed and placed in the thermal sequencer. After amplification, the tips are removed and the contents loaded directly onto an agarose gel. Reagent consumption is less, cycling time is decreased and direct loading of samples is an advantage. It is not easy to predict future developments in nucleic acids research in environmental microbiology/biotechnology. Most methods currently in use for directly extracting DNA from soil, sediment and water require small samples (0.5-1.0g fresh soil/sediment or 1 ml water). Moreover, water samples can be filtered (if not too turbid) to concentrate cell numbers and, therefore, improve detection
limits for cells. Soil and sediment extracts can be purified using spun-columns (e.g. Sephadex G50 or G75) and glass milk clean-up protocols that yield D N A suitable as a template for PCR analysis [8]. Because most methods use small samples, a large number of samples can be processed in a short period of time in microfuge tubes. This allows researchers to increase the number of samples in their experiment without a pronounced increase in the amount of time and effort. This is especially important in environmental microbiology/biotechnology where a complex matrix, such as soil or sediment, may not have the target PCR sequence or microorganism randomly distributed throughout the experimental system. By increasing the number of replicates 'n', the researcher is able to obtain more representative data. One immediate concern in environmental research is the sensitive detection and monitoring of released genetically engineered microorganisms (GEMs) in the environment over months to years. PCR methodology provides limits of detection in the range of 1 to 100 G E M s per gram of soil or sediment [21. PCR-based methods will continue to gain importance as an alternative or amendment to conventional standard methods used in public health microbiology. T h e reader is also referred to an excellent chapter by Torsvik et al. [9°'], who review the use of DNA analysis (DNA reassociation analysis) to determine the diversity of microbial communities in soil from Norway. These researchers have shown that the genetic diversity (measure of the number and frequency of genetically diverse microorganisms in a population or community) of soil microbial communities is -200-fold higher than the diversity of the isolated bacteria, and therefore the community probably consists of thousands of species. A DNA reassociation assay involves comparison of total genomic DNA between bacteria. Therefore, it is a good measurement of genetic relatedness between different species. T h e rate of DNA reassociation is an inverse function of genomic c o m p l e x i t y - - t h e total number of nucleotide pairs in non-repeating sequences. Normally, DNA reassociation follows second-order kinetics, and a half association value (Cotl/2) is used to express genomic complexity. It is defined by the following equation:
C/C0
=
l ff l +kC~¢)
where t is time, C is the concentration of single-stranded DNA at time t, CO is the concentration of single-stranded DNA at t= 0 and k is the DNA reassociation rate constant. When C= 1/2(Co), Cotl/2 = 1/k, where tl/2 is the time for half of the DNA fragments to reassociate.
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Reporter genes In 1994, an excellent review on molecular marker systems (reporter genes) for the detection of GEMs in the environment was published [10"]. T h e use of unique DNA sequences for the detection of GEMs released into the environment will assist in the risk-assessment process and ensure the safe use of environmental biotechnology. T h e primary reporter genes used to date are antibiotic resistance genes, lacZY, xylE and lux. Antimicrobial resistance markers (e.g. kanamycin, tetracycline, ampicillin, rifampicin and nalidixic acid) permit selection of viable populations resistant to the marker(s) by plating on a suitable agar medium containing the selective antibiotic(s). There is, however, concern over the use of antimicrobial resistance genes and their contribution to the resistance gene pool in nature. Therefore, more suitable markers, such as lacZY (which encode I]-galactosidase and lactose permease), are often used. Microorganisms expressing these genes can be detected on solid medium containing the chromogenic X-Gal (5-chloro-4-bromo-3indolyl I]-D-galactopyranoside). When X-Gal is enzymatically cleaved, it produces blue-green colonies that can be detected by the naked eye and enumerated. This system is useful only for microorganisms that normally do not utilize lactose (e.g. Pseudomonas spp.) [11,12]. T h e lacZY reporter system can be used in conjunction with a bioluminescence (lux)-based marker system. Bioluminescence genes luxABCDE were originally cloned from luminescent marine bacteria Vibrio fischeri or Vibrio harveyi, and they encode enzymes responsible for light emission when long-chain fatty aldehyde substrates are oxidized [10"]. Bioluminescence is normally not found in soil bacteria. Bacterial luminescence results from luciferase enzyme activity (encoded by structural genes luxA and luxB) in the presence of oxygen and a substrate such as n-decanal (or normally n-tetradecyl aldehyde) and reducing equivalents [10°]. Light-emitting colonies can be easily detected using a Biological Image Quantifier (Image Research Ltd. Cambridge, UK) [8,11-13], or X-ray/photographic film [10"]. This system is very useful for detection of bacterial cells in contaminated soil and sediment [12]. Another marker system is the xylE gene (which encodes catechol 2,3-dioxygenase). This gene can be cloned into a recombinant plasmid to allow rapid colony screening for microorganisms introduced into the environment using non-selective media [10°]. After being sprayed with a 1% solution of catechol, bacterial colonies carrying this marker gene produce a yellow colour resulting from the formation of hydroxymuconic semialdehyde. Positive colonies can be confirmed using immunological or genetic techniques.
Natural genetic transformation For an excellent discussion of bacterial gene transfer in the environment, the reader is referred to the 1994 review by Lorenz and Wackernagel [14"]. In environmental microbiology/biotechnology, there is still a paucity of
research on both the persistence of nucleic acids in soil, water and sediment and their role in genetic transformation in these environments. Natural genetic transformation is the active uptake by competent microbial cells of extracellular (free) DNA (plasmid or chromosomal) followed by the incorporation of its genetic information [14",15]. Natural genetic transformation differs from genetic conjugation and transduction by its sensitivity to DNases [14"]. Also, genetic transformation does not require a living donor cell(s). The release of DNA during and after the death of microbial cells can provide DNA for genetic transformation. Even so, the recipient microbial cells must be capable of taking up the DNA. If adsorption of DNA to soil particles protects DNA from DNase digestion, genetic transformation of microbial cells may still occur. Recently, the binding of DNA (from Pseudomonas stutzeH Zobell PP101) to sterile marine sediment in the presence of divalent cations (Mg2+, Ca2+ and Mn2+) has been investigated [16], and its effects on natural genetic transformation of P stutzeri strain Zobell studied [17]. DNA binding to sterile sediment in the presence of varying concentrations of cations was observed to protect DNA from DNase digestion. Moreover, the binding was shown to decrease the frequency of natural transformation.
DNA adsorption to soil Following adsorption to clays, nucleic acids can form organo-mineral complexes resistant to digestion by nucleases. Competence is the ability of bacterial cells to bind and take up DNA in a form resistant to intracellular digestion by DNases. DNA entry occurs at a limited number of receptor sites on the surface of the bacterial cells. Passage of DNA across the cell wall and membrane requires both energy and specific transport molecules. This has been determined by the fact that inhibition of protein synthesis and/or energy production in recipient cells inhibits genetic transformation. For DNA to be transformed efficiently, it should be in the size range 10-20kbp (i.e. a fragment capable of encoding several microbial genes). In a recent study, Ogram et al. [18] have confirmed that the adsorption of DNA to different soils follows the Freundlich adsorption model. Smaller DNA fragments (2.69kbp or 1.75MDa) are sorbed to soils better than larger DNA fragments (23 kbp or 14.95 MDa) [19].
Conclusions T h e preceding year has seen excellent progress in the application of nucleic acids based techniques in environmental microbiology/biotechnology. This is demonstrated by the expanding use of direct extraction of DNA/RNA from environmental samples and detection of specific DNA sequences. These applications can assist environmental microbiology problems such as the detection of
Nucleic acids in the environment Trevors
specific microorganisms that were previously undetectable or difficult/time-consuming to detect as a result of the limitations in conventional methods (e.g. viable counts on plates). Additional research will also likely be forthcoming on new methods for purifying DNA and RNA from soil, sediment and water samples. Currently, soil and sediment samples can be as small as 0.5-1.0 g. There may be some additional refinements to minimum samples sizes used and automation of sample handling to allow processing of hundreds of samples at one time. Some improvements will also probably be made in the direct extraction of DNA. For example, the detachment of microbial cells from soils such as heavy clays or soils high in organic matter and the lysis of microbial cells in the interior of soil aggregates could be improved by the introduction of more effective approaches. This will facilitate extraction of a more representative sample of the microbial community and improve detection limits for the target D N A sequence(s). T h e past year also brought forth research chapters on the extraction of RNA from deep marine sediments, the ability of naked DNA in marine sediment to genetically transform P. stutzeH and DNA adsorption to soil. Natural genetic transformation differs from genetic conjugation and transduction by its sensitivity to DNases and the fact that transformation does not require a living donor cell(s). T h e release of D N A during and after the death of microbial cells can provide sufficient DNA for genetic transformation. However, the recipient microbial cells must be capable of taking up the DNA. If adsorption of DNA to soil particles protects DNA from DNase digestion, genetic transformation of microbial cells may still occur. With respect to reporter gene technology, it is likely that new reporter genes will be constructed in the future. For example, Cebolla et al. [19] have recently reported on expression vectors carrying eukaryotic luciferases as markers, each luminescing with a characteristic color. These would permit the distinction of two populations of the same or different microorganisms on the basis of their color or luminescence. Another potentially useful reporter for use in environmental microbiology/biotechnology research is the green fluorescent protein (GFP) [20"°], a naturally fluorescent protein from a jellyfish, Aequorea victoria. (GFP is available from Clontech, 4030 Fabian Way, Palo Alto, California 94303-9605, USA.) Bacterial colonies containing this cloned gene can be detected under illumination from long-wave ultraviolet light. T h e GFP fluoresces in the absence of any other proteins, substrates or co-factors. Little information exists on the use of the G F P as a reporter gene in microorganisms in soil, sediment and water.
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T h e future will be exciting for those participating in research on nucleic acids in the environment. This was demonstrated in 1995 by the publication of two laboratory manuals, Molecular Microbial Ecology Manual [21"] and Nucleic Acids in the Environment [4°]. Both texts have a strong emphasis on the use of molecular based nucleic acids techniques in soil, rhizosphere, sediment and water.
Acknowledgements The financial assistance of the Natural Sciences and Engineering Research Council of Canada (NSERC) operating grants program is gratefully acknowledged.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest =* of outstanding interest Selenska S, Klingmuller W: Direct recovery and molecular analysis of DNA and RNA from soil. Microb Releases 1992, 1:41-46. 2.
Bej AK, Mahbubani MH: Applications of the polymerase chain reaction (PCR) in vitro DNA-amplificatlon method in environmental microbiology. In PCR Technology: Current Innovations. Edited by Griffin HG, Griffin AM. Boca Raton: CRC Press; 1994:327-339.
3.
Griffin HG, Griffin AM (Eds): PCR Technology: Current Innovations. Boca Raton: CRC Press; 1994.
TrevorsJT, Van Elsas JD (Eds): Nucleic Acids in the Environment: Methods and Applications. Heidelberg: Springer-Verlag; 1995. his text contains theory and methods for extraction, purification and amplification of nucleic acids from environmental samples. Topics discussed include recovery of bacterial cells from soil, direct extraction of DNA from soil, extraction and amplification of DNA from the rhizosphere and rhizoplane of plants, extraction of DNA from the phyllosphere, specific DNA sequences for the detection of soil bacteria, PCR amplification of DNA from root nodules, extraction of DNA/RNA from aquatic environments, PCR amplification of DNA recovered from the aquatic environment, extraction and amplification of 16S rRNA genes from deep marine sediments and PCR for detection of antibiotic resistance genes in environmental samples. 4.
Tsai YL, Park MJ, Olson BH: Rapid method for direct extraction of mRNA from seeded soils. Appl Environ Microbiol 1991, 57:765-768. 6. •
Pichard SL, Paul JH: Detection of gene expression in genetically engineered microorganisms and natural phytoplankton populations in the marine environment by mRNA analysis, App/Environ Microbio/1994, 57:1721-1727. A useful manuscript that describes the extraction of mRNA from aquatic samples, allowing an analysis of gene expression. Torsvik V, Daae FL, Goksoyr J: Extraction, purification and analysis of DNA from soil bacteria. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:29-48. Leung K, Trevors JT, Van Elsas JD: Extraction and amplification of DNA from the rhizosphere and rhizoplane of plants. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:69-87. 9. •e
TorsvikV, Goksoyr J, Daae FL, Sorheim R, Michalsen J, Salte K: Use of DNA analysis to determine the diversity of microbial communities. In Beyond the Biomass. Edited by Ritz K, Dighton J, Giller KE. London: British Society of Soil Science/Wiley.Sayce Publications; 1994:39-48. An excellent paper on DNA diversity in soil microbial populations on the basis of DNA reassociation of highly purified microbial DNA from soil microorganisms. The diversity of the total soil microbial community is in the order of thousands of species.
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Prosser Jl: Molecular marker systems for detection of genetically engineered micro-organisms in the environment. Microbiology 1994, 140:5-17. This is an excellent review of marker/reporter genes for use in the environment.
of microbial nucleic acids, detection of microbial nucleic acid sequences, identification and classification of microbes using DNA and RNA sequences, detection of gene transfer in the environment, tracking of specific microbes in the environment, and designing field and microcosm experiments with GEMs.
11.
Flemming CA, Leung KT, Lee H, Trevors JT, Greet C: Survival of a lux-lac marked biosurfactant-producing Pseudomonas aeruginosa UG2 strain in soil: monitored by non-selective plating and PCR techniques. App/Environ Micmbiol 1994, 60:1606-1613.
22.
Saano A, Tas E, Pippola S, Lindstrom K, Van Elsas JD: Extraction and analysis of microbial DNA from soil. In Nucleic Acids in the Environment: Methods and Appfications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vedag; 1995:49-67.
23.
12.
Van Dyke MI, Lee H, Trevors JT: Survival and polymerase chain reaction detection of luxAB-marked Alcaligenes eutrophus H850 in PCB-contaminated soil and sediment in the presence of rhamnolipid biosurfactants. J Chem Technol Biotechno/1996, 65:115-122.
Bailey MJ: Extraction of DNA from the phyllosphere. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors iT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:89-109.
24.
NesmeX, Picard C, Simonet P: Specific DNA sequences for detection of soil bacteria. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vedag; 1995:112-139.
25.
Pepper IL, Pillai SD: PCR amplification of DNA from root nodules. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:141-151.
26.
Paul JH, Pichard SL: Extraction of DNA end RNA from aquatic environments. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors iT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:154-1 77.
2?.
Bej AK: PCR amplification of DNA recovered from the aquatic environment. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vertag; 1995:179-218.
28.
Rochelle PA, Will JAK, Fry JC, Jenkins GJS, Parkes RJ, Turley CM, Weightman AJ: Extraction and amplification of 16S rRNA genes from deep marine sediments and seawater to assess bacterial community diversity. In Nucleic Acids in the Environment: Methods and Appfications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vertag; 1995:119-239.
29.
Smalla K, Van Elsas JD: Application of the PCR for detection of antibiotic resistance genes in environmental samples. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:242-256.
30.
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 1994, 263:802-805. The description of GFP as a marker reporter gene for use in biological research. This is an excellent paper that has stimulated a considerable amount of research on the use of GFP as a marker gene.
Pepper IL, Pillai SD: Detection of specific DNA sequences in environmental samples via polymerase chain reaction. In Methods of Soil Analysis Part 2: Microbiological and Biochemical Properties. Madison: Soil Science Society of America; 1994:707-726.
31.
Holben WE: Isolation and purification of bacterial DNA from soil. In Methods of Soil Analysis Part 2: Microbiological and Biochemical Properties. Madison: Soil Science Society of America; 1994:727-751.
21. •
32.
Ryder M: Key issues in the deliberate release of geneticallymanipulated bacteria. FEMS Microbiol Ecol 1994, 15:139-146.
33.
TrevorsJ: Molecular evolution in bacteria. Antonie Van Leeuwenhoek 1995, 67:315-324.
10.
13.
Leung K, Cassidy M, Holmes, Lee H, Trevors JT: Survival of carrageenan-ensapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr in a forest soil: monitored by polymerase chain reaction and viable plating. FEMS Microbiol Eco/ 1995,16:71-82.
14. •
Lorenz MG, Wackernagel W: Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 1994, 58:563-602. A review on natural genetic transformation in the environment as a mechanism of gene transfer or gene flow. The authors provide an excellent description of the fundamentals of natural genetic transformation and the bacterial species involved.
15.
TrevorsJT: DNA in soil: adsorption, genetic transformation and molecular evolution. Antonie van Leeuwenhoek 1996, in press.
16.
Stewart G J, Garko KA: The effect of divalent cations on the frequency of natural transformation in Pseudomonas stutzeri strain Zobell in marine sediment. Microb Releases 1994, 2:201-207.
1 7.
Garko KA, Stewart GJ: The effect of divalent cations on the binding of divalent cations to marine sediment. Microb Releases 1994, 2:191-199.
18.
Ogram AV, Mathot ML, Harsh JB, Boyle J, Pettigrew CA Jr: Effects of DNA polymer length on its adsorption to soils. Appl Environ Microbiol 1994, 60:393-396.
19.
Cebolla A, Vazquez ME, Palomares AJ: Expression vectors for the use of eukaryotic luciferases with different colors of luminescence. App/ Environ Microbio/1995, 61:660-668.
20. •-
AkkermansADL, Van Elsas JD, De Bruijn FJ (Eds): Molecular Microbial Ecology Manual. Dordrecht: Kluwer Academic Publishers; 1995. This comprehensive manual contains molecular protocols relevant in microbial ecology research. The manual is divided into seven sections: isolation
Nucleic acids in the environment Jack T Trevors The past year has witnessed several excellent advances in basic and applied research on nucleic acids in the environment. Improved methods for extracting nucleic acids from environmental samples have been published, as well as information on the use of reporter genes in bacteria, natural genetic transformation in soil and DNA adsorption to soil. These advances will have a significant impact on both future research and the way in which we view nucleic acids in the environment.
Address Laboratory of Microbial Technology, Department of Environmental Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1; e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:331-336 © Current Biology Ltd ISSN 0958-1669 Abbreviations GEM geneticallyengineered microorganism GFP green fluorescent protein
Introduction
Nucleic acids (i.e. DNA and RNA) are virtually ubiquitous in the biosphere. If you look past the soil, water, sediment, vegetation and animals on the Earth, and imagine the amount of DNA necessary to encode for the multitude of living organisms, an appreciation of the amount of D N A and RNA in the environment can be obtained. For example, Selenska and Klingmuller [1] reported that - 3 0 0 n g DNA and - 1 0 0 n g RNA can be extracted from 10g soil. This value does not include DNA/RNA in the rhizosphere, other plant-derived DNA/RNA or DNA/RNA from organisms above the soil surface. T h e Earth is virtually covered with nucleic acids. Not only is the amount of DNA on the Earth overwhelming, but its diversity and ability to undergo genetic recombination and mutation while retaining a degree of constancy illustrate both the complexity of life on this planet and the evolutionary path(s) that have brought life on Earth to its present state. Researchers and students of environmental microbiology (and other scientific disciplines) are poised at the beginning of an era in which the use of molecular biology in their field is still in its infancy. For example, the use of PCR in environmental microbiology was reviewed in 1994 by Bej and Mahbubani [2]. T h e high specificity, sensitivity and reproducible consistency of PCR detection of specific DNA sequences in complex environmental samples has contributed significantly to the advancement of knowledge in environmental microbiology and many other areas of research (e.g. detection of food-borne microbial pathogens, clinical diagnostics, forensic science, genome mapping, plant taxonomy and evolution, detec-
tion of mutations, mutagenesis by PCR, generation of DNA probes by PCR, and the cloning of PCR products) [3]. T h e past year has seen numerous improvements to, and applications of, PCR in environmental microbiology, especially with soil, sediment, water and plant material. A recently published text [4"] includes information on the theory and protocols for the extraction of DNA and RNA from environmental samples and covers developments from the past 15 years. T h e majority of the research and methods has, however, originated in the past 8 years. This review examines recent advances in nucleic acids in the environment from an environmental microbiology/biotechnology perspective. To date, most of the research has focused on DNA, with less emphasis on RNA extraction and purification. Yet mRNA can now be extracted from environmental samples such as soil [5] and sediment [6"]. Although these methods were introduced before the past year, they are significant to the area of nucleic acids in the environment and, therefore, are cited here. Other research discussed in this review includes reporter gene technology, genetic transformation in the environment and DNA adsorption to soil (see Table 1). Direct DNA extraction
and PCR amplification
Direct soil DNA extraction techniques are now an important part of microbial ecology research. These methods are useful in the investigation of indigenous bacteria or bacteria introduced into the environment. Because viable microbial cells can be only partially recovered from complex environmental samples by traditional viable plating methods, cell extraction from soil and sediment samples recovers only a fraction of the cells present. Also, a large fraction, often 90-99%, of microbial cells present in environmental samples are not culturable on microbiological media and, therefore, escape detection when cultivation-based methods are used. Because a large percentage of microbial cells in environmental samples may be viable but non-culturable, their detection via molecular techniques is often necessary. Only in recent years has microbial DNA in the environment been studied using the powerful technology of PCR. Complex environmental samples, such as soil, sediment and water, from which D N A (or RNA) is extracted contain a multitude of substances (e.g. clays, metals, toxic chemicals, nucleases and humic material) that can inhibit the PCR reaction. For this reason, environmental samples must be highly purified to be used as templates in PCR analysis. It is now possible to obtain highly purified DNA from environmental samples in short periods of time (several hours) that can be used for PCR analysis. T h e
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Environmental biotechnology
Table 1
Figure 1
Some recent research on nucleic acids In the environmenL
Research Direct extraction and PCR Extraction, purification and analysis of DNA from soil bacteria Extraction and amplification of DNA from rhizophere and rhizoplane Extraction of DNA from phyllosphere Specific DNA sequences for detection of soil bacteria PCR amplification of DNA from root nodules Extraction of DNA/RNA from aquatic environments PCR amplification of DNA from aquatic environments Extraction and amplification of 16S rRNA genes from deep marine sediments and seawater PCR for detection of antibiotic resistance genes in environmental samples DNA analysis to determine diversity of microbial communities Detection of DNA sequences in environmental samples by PCR Isolation, purification of bacterial DNA from soil Reporter genes Survival of lux-lac marked P. aeruginosa UG2 in soil monitored by PCR Survival of carrageenan-encapsulated and unencapsulated P. aeruginosa UG2Lr in forest soil monitored by PCR and viable plating Molecular marker systems for detecting GEMs in the environment PCR detection of luxAB-marked Alcaligenes eutrophus H850 polychlorinated biphenyl contaminated soil and sediment Natural transformation Effect of divalent cations on natural transformation of P. stutzeri in marine sediment DNA in soil: adsorption, genetic transformation and molecular evolution Bacterial gene transfer by natural genetic transformation in the environment DNA adsorption to soil Effects of DNA polymer length on its adsorption to soils Molecular evolution In bacteria Information on molecular evolution of bacteria, including microbiology, molecular biology, metal-microbe interactions and restrictionmodification systems
References [22] [8] [23] [24] [25] [26] [27] [28] [29] [9"] [30] [31] [11 ] [13] [10",32] [12]
[8] [23]
Generalized procedure for direct DNA extraction from soil or sediment. One advantage of this procedure is that only a small sample (0.5-1.0g) is required and the DNA is extracted directly from the sample without first extracting the microbial cells from the sample. It is necessary to achieve good lysis of the microbial cells in samples to ensure the DNA under study is released and recovered. This is especially important if the number of cells containing the DNA of interest is low. Once the cells are lysed, the extract can be subjected to additional purification procedures using a spun column and glass-milk clean-up procedures. The recovered DNA can then be quantified and used for PCR analysis or other laboratory manipulations.
[24]
[181
[33]
ability to obtain amplifiable D N A from D N A extracts directly is significant given that the ability to recovery bacterial cells from soil can range from about 7% to 18% [7] and often is lower. Figure 1 summarizes the main steps in the direct extraction of DNA from soil or sediment. Most methods add phosphate or Tris buffers to soil, followed by rapid mixing (several hundred to thousands of rpm) for -5-30rain at room temperature to initially produce a slurry in which subsequent lysis of cells followed by
extraction and purification of DNA can be achieved. Some methods use repeated freezing (--80"C) and thawing (room temperature or higher) cycles to achieve lysis of the microbial cells. T h e extract obtained is generally sufficiently pure to use as a template in PCR reactions. T h e additional purification using glass milk (Geneclean II, Bio 101 Inc, La Jolla, California, USA) reduces the DNA yield, but provides highly purified DNA that can be used as a D N A template. One recent approach is the use of magnetic beads (Dynabeads M-280 streptavidin, Dynal Inc, Lake Success, New York, USA) linked to a single-stranded specific DNA probe for selective recovery of complementary target sequences from samples, which are subsequently amplified for detection and/or isolation. Nucleic acids obtained from complex environmental samples should ideally be representative of the biota present. With these samples, it is also necessary to assess both the quality and quantity of the nucleic acids obtained. T h e quantity of extracted and purified DNA can be assessed on agarose gels and compared with DNA size ladders.
Nucleic acids in the environment Trevors 333
DNA quantification based on spectrophotometric analysis at 260nm does not work well for soil-derived D N A because of the presence of strongly absorbing compounds in soil. Alternative methods include fiuorometric determination of DNA using Hoechst dye 33258 (bisbenzimide: 2-[2-(4-hydroxyphenyl)-6-benzimidazoly)]6-(1-methyl-4-piperazyl) benzmidazole-3HCl pentahydrate [Calbiochem Diagnostics, La Jolla, California, USA]) and ethidium bromide (2,7-diamino-10-ethyly9-phenyl-phenanthridinium bromide [8]. Fluorescence measurements using Hoechst 33258 can be measured using a Hoefer 100 Mini-fluorometer (Hoefer Scientific Instruments, San Francisco, California, USA) calibrated with purified EscheHchia coli DNA (Sigma Chemical Company, St Louis, USA). DNA concentrations in the nanogram range can be quantified. A 2 nl sample is adequate for the determination; therefore, most of the sample is available for other analyses. DNA concentrations are expressed as nanogram per gram of dry soil/sediment/root or nanogram per milliliter of water. Another fluorometric method for quantification of D N A employs ethidium bromide as the fluorescent dye and a Tyler Model SSF-600 solid-state fluorometer (Tyler Research, Edmonton, Alberta, Canada) to measure fluorescence [81. Ethidium bromide absorbs light at both 280 nm and 520 nm, with excitation of either wavelength giving rise to emission at 600nm. T h e SSF-600 excites at 520nm and measures the emission at 600nm. A third method for assessing the DNA content of soil is based on scanning (using a Pharmacia LKB UltroScan XL Scanner) of pictures of ethidium bromide stained DNA bands in agarose gels and their comparison with pure D N A standards. This method has the advantage of electrophoretic removal of interference by humic material (electrophoresed DNA is highly purified). It is also possible to use the ability to successfully digest the DNA with restriction enzymes or the ability to amplify a target sequence by PCR, as measures of DNA purity. With respect to thermal-cycling equipment, a fast capillary thermal sequencer (FTS-IS; Corbett Research, New South Wales, Australia) can achieve 30 cycles in 30 min and does not require an oil overlay. This permits direct loading of reagents and samples in a capillary tube, which is sealed and placed in the thermal sequencer. After amplification, the tips are removed and the contents loaded directly onto an agarose gel. Reagent consumption is less, cycling time is decreased and direct loading of samples is an advantage. It is not easy to predict future developments in nucleic acids research in environmental microbiology/biotechnology. Most methods currently in use for directly extracting DNA from soil, sediment and water require small samples (0.5-1.0g fresh soil/sediment or 1 ml water). Moreover, water samples can be filtered (if not too turbid) to concentrate cell numbers and, therefore, improve detection
limits for cells. Soil and sediment extracts can be purified using spun-columns (e.g. Sephadex G50 or G75) and glass milk clean-up protocols that yield D N A suitable as a template for PCR analysis [8]. Because most methods use small samples, a large number of samples can be processed in a short period of time in microfuge tubes. This allows researchers to increase the number of samples in their experiment without a pronounced increase in the amount of time and effort. This is especially important in environmental microbiology/biotechnology where a complex matrix, such as soil or sediment, may not have the target PCR sequence or microorganism randomly distributed throughout the experimental system. By increasing the number of replicates 'n', the researcher is able to obtain more representative data. One immediate concern in environmental research is the sensitive detection and monitoring of released genetically engineered microorganisms (GEMs) in the environment over months to years. PCR methodology provides limits of detection in the range of 1 to 100 G E M s per gram of soil or sediment [21. PCR-based methods will continue to gain importance as an alternative or amendment to conventional standard methods used in public health microbiology. T h e reader is also referred to an excellent chapter by Torsvik et al. [9°'], who review the use of DNA analysis (DNA reassociation analysis) to determine the diversity of microbial communities in soil from Norway. These researchers have shown that the genetic diversity (measure of the number and frequency of genetically diverse microorganisms in a population or community) of soil microbial communities is -200-fold higher than the diversity of the isolated bacteria, and therefore the community probably consists of thousands of species. A DNA reassociation assay involves comparison of total genomic DNA between bacteria. Therefore, it is a good measurement of genetic relatedness between different species. T h e rate of DNA reassociation is an inverse function of genomic c o m p l e x i t y - - t h e total number of nucleotide pairs in non-repeating sequences. Normally, DNA reassociation follows second-order kinetics, and a half association value (Cotl/2) is used to express genomic complexity. It is defined by the following equation:
C/C0
=
l ff l +kC~¢)
where t is time, C is the concentration of single-stranded DNA at time t, CO is the concentration of single-stranded DNA at t= 0 and k is the DNA reassociation rate constant. When C= 1/2(Co), Cotl/2 = 1/k, where tl/2 is the time for half of the DNA fragments to reassociate.
334 Environmentalbiotechnology
Reporter genes In 1994, an excellent review on molecular marker systems (reporter genes) for the detection of GEMs in the environment was published [10"]. T h e use of unique DNA sequences for the detection of GEMs released into the environment will assist in the risk-assessment process and ensure the safe use of environmental biotechnology. T h e primary reporter genes used to date are antibiotic resistance genes, lacZY, xylE and lux. Antimicrobial resistance markers (e.g. kanamycin, tetracycline, ampicillin, rifampicin and nalidixic acid) permit selection of viable populations resistant to the marker(s) by plating on a suitable agar medium containing the selective antibiotic(s). There is, however, concern over the use of antimicrobial resistance genes and their contribution to the resistance gene pool in nature. Therefore, more suitable markers, such as lacZY (which encode I]-galactosidase and lactose permease), are often used. Microorganisms expressing these genes can be detected on solid medium containing the chromogenic X-Gal (5-chloro-4-bromo-3indolyl I]-D-galactopyranoside). When X-Gal is enzymatically cleaved, it produces blue-green colonies that can be detected by the naked eye and enumerated. This system is useful only for microorganisms that normally do not utilize lactose (e.g. Pseudomonas spp.) [11,12]. T h e lacZY reporter system can be used in conjunction with a bioluminescence (lux)-based marker system. Bioluminescence genes luxABCDE were originally cloned from luminescent marine bacteria Vibrio fischeri or Vibrio harveyi, and they encode enzymes responsible for light emission when long-chain fatty aldehyde substrates are oxidized [10"]. Bioluminescence is normally not found in soil bacteria. Bacterial luminescence results from luciferase enzyme activity (encoded by structural genes luxA and luxB) in the presence of oxygen and a substrate such as n-decanal (or normally n-tetradecyl aldehyde) and reducing equivalents [10°]. Light-emitting colonies can be easily detected using a Biological Image Quantifier (Image Research Ltd. Cambridge, UK) [8,11-13], or X-ray/photographic film [10"]. This system is very useful for detection of bacterial cells in contaminated soil and sediment [12]. Another marker system is the xylE gene (which encodes catechol 2,3-dioxygenase). This gene can be cloned into a recombinant plasmid to allow rapid colony screening for microorganisms introduced into the environment using non-selective media [10°]. After being sprayed with a 1% solution of catechol, bacterial colonies carrying this marker gene produce a yellow colour resulting from the formation of hydroxymuconic semialdehyde. Positive colonies can be confirmed using immunological or genetic techniques.
Natural genetic transformation For an excellent discussion of bacterial gene transfer in the environment, the reader is referred to the 1994 review by Lorenz and Wackernagel [14"]. In environmental microbiology/biotechnology, there is still a paucity of
research on both the persistence of nucleic acids in soil, water and sediment and their role in genetic transformation in these environments. Natural genetic transformation is the active uptake by competent microbial cells of extracellular (free) DNA (plasmid or chromosomal) followed by the incorporation of its genetic information [14",15]. Natural genetic transformation differs from genetic conjugation and transduction by its sensitivity to DNases [14"]. Also, genetic transformation does not require a living donor cell(s). The release of DNA during and after the death of microbial cells can provide DNA for genetic transformation. Even so, the recipient microbial cells must be capable of taking up the DNA. If adsorption of DNA to soil particles protects DNA from DNase digestion, genetic transformation of microbial cells may still occur. Recently, the binding of DNA (from Pseudomonas stutzeH Zobell PP101) to sterile marine sediment in the presence of divalent cations (Mg2+, Ca2+ and Mn2+) has been investigated [16], and its effects on natural genetic transformation of P stutzeri strain Zobell studied [17]. DNA binding to sterile sediment in the presence of varying concentrations of cations was observed to protect DNA from DNase digestion. Moreover, the binding was shown to decrease the frequency of natural transformation.
DNA adsorption to soil Following adsorption to clays, nucleic acids can form organo-mineral complexes resistant to digestion by nucleases. Competence is the ability of bacterial cells to bind and take up DNA in a form resistant to intracellular digestion by DNases. DNA entry occurs at a limited number of receptor sites on the surface of the bacterial cells. Passage of DNA across the cell wall and membrane requires both energy and specific transport molecules. This has been determined by the fact that inhibition of protein synthesis and/or energy production in recipient cells inhibits genetic transformation. For DNA to be transformed efficiently, it should be in the size range 10-20kbp (i.e. a fragment capable of encoding several microbial genes). In a recent study, Ogram et al. [18] have confirmed that the adsorption of DNA to different soils follows the Freundlich adsorption model. Smaller DNA fragments (2.69kbp or 1.75MDa) are sorbed to soils better than larger DNA fragments (23 kbp or 14.95 MDa) [19].
Conclusions T h e preceding year has seen excellent progress in the application of nucleic acids based techniques in environmental microbiology/biotechnology. This is demonstrated by the expanding use of direct extraction of DNA/RNA from environmental samples and detection of specific DNA sequences. These applications can assist environmental microbiology problems such as the detection of
Nucleic acids in the environment Trevors
specific microorganisms that were previously undetectable or difficult/time-consuming to detect as a result of the limitations in conventional methods (e.g. viable counts on plates). Additional research will also likely be forthcoming on new methods for purifying DNA and RNA from soil, sediment and water samples. Currently, soil and sediment samples can be as small as 0.5-1.0 g. There may be some additional refinements to minimum samples sizes used and automation of sample handling to allow processing of hundreds of samples at one time. Some improvements will also probably be made in the direct extraction of DNA. For example, the detachment of microbial cells from soils such as heavy clays or soils high in organic matter and the lysis of microbial cells in the interior of soil aggregates could be improved by the introduction of more effective approaches. This will facilitate extraction of a more representative sample of the microbial community and improve detection limits for the target D N A sequence(s). T h e past year also brought forth research chapters on the extraction of RNA from deep marine sediments, the ability of naked DNA in marine sediment to genetically transform P. stutzeH and DNA adsorption to soil. Natural genetic transformation differs from genetic conjugation and transduction by its sensitivity to DNases and the fact that transformation does not require a living donor cell(s). T h e release of D N A during and after the death of microbial cells can provide sufficient DNA for genetic transformation. However, the recipient microbial cells must be capable of taking up the DNA. If adsorption of DNA to soil particles protects DNA from DNase digestion, genetic transformation of microbial cells may still occur. With respect to reporter gene technology, it is likely that new reporter genes will be constructed in the future. For example, Cebolla et al. [19] have recently reported on expression vectors carrying eukaryotic luciferases as markers, each luminescing with a characteristic color. These would permit the distinction of two populations of the same or different microorganisms on the basis of their color or luminescence. Another potentially useful reporter for use in environmental microbiology/biotechnology research is the green fluorescent protein (GFP) [20"°], a naturally fluorescent protein from a jellyfish, Aequorea victoria. (GFP is available from Clontech, 4030 Fabian Way, Palo Alto, California 94303-9605, USA.) Bacterial colonies containing this cloned gene can be detected under illumination from long-wave ultraviolet light. T h e GFP fluoresces in the absence of any other proteins, substrates or co-factors. Little information exists on the use of the G F P as a reporter gene in microorganisms in soil, sediment and water.
335
T h e future will be exciting for those participating in research on nucleic acids in the environment. This was demonstrated in 1995 by the publication of two laboratory manuals, Molecular Microbial Ecology Manual [21"] and Nucleic Acids in the Environment [4°]. Both texts have a strong emphasis on the use of molecular based nucleic acids techniques in soil, rhizosphere, sediment and water.
Acknowledgements The financial assistance of the Natural Sciences and Engineering Research Council of Canada (NSERC) operating grants program is gratefully acknowledged.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest =* of outstanding interest Selenska S, Klingmuller W: Direct recovery and molecular analysis of DNA and RNA from soil. Microb Releases 1992, 1:41-46. 2.
Bej AK, Mahbubani MH: Applications of the polymerase chain reaction (PCR) in vitro DNA-amplificatlon method in environmental microbiology. In PCR Technology: Current Innovations. Edited by Griffin HG, Griffin AM. Boca Raton: CRC Press; 1994:327-339.
3.
Griffin HG, Griffin AM (Eds): PCR Technology: Current Innovations. Boca Raton: CRC Press; 1994.
TrevorsJT, Van Elsas JD (Eds): Nucleic Acids in the Environment: Methods and Applications. Heidelberg: Springer-Verlag; 1995. his text contains theory and methods for extraction, purification and amplification of nucleic acids from environmental samples. Topics discussed include recovery of bacterial cells from soil, direct extraction of DNA from soil, extraction and amplification of DNA from the rhizosphere and rhizoplane of plants, extraction of DNA from the phyllosphere, specific DNA sequences for the detection of soil bacteria, PCR amplification of DNA from root nodules, extraction of DNA/RNA from aquatic environments, PCR amplification of DNA recovered from the aquatic environment, extraction and amplification of 16S rRNA genes from deep marine sediments and PCR for detection of antibiotic resistance genes in environmental samples. 4.
Tsai YL, Park MJ, Olson BH: Rapid method for direct extraction of mRNA from seeded soils. Appl Environ Microbiol 1991, 57:765-768. 6. •
Pichard SL, Paul JH: Detection of gene expression in genetically engineered microorganisms and natural phytoplankton populations in the marine environment by mRNA analysis, App/Environ Microbio/1994, 57:1721-1727. A useful manuscript that describes the extraction of mRNA from aquatic samples, allowing an analysis of gene expression. Torsvik V, Daae FL, Goksoyr J: Extraction, purification and analysis of DNA from soil bacteria. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:29-48. Leung K, Trevors JT, Van Elsas JD: Extraction and amplification of DNA from the rhizosphere and rhizoplane of plants. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:69-87. 9. •e
TorsvikV, Goksoyr J, Daae FL, Sorheim R, Michalsen J, Salte K: Use of DNA analysis to determine the diversity of microbial communities. In Beyond the Biomass. Edited by Ritz K, Dighton J, Giller KE. London: British Society of Soil Science/Wiley.Sayce Publications; 1994:39-48. An excellent paper on DNA diversity in soil microbial populations on the basis of DNA reassociation of highly purified microbial DNA from soil microorganisms. The diversity of the total soil microbial community is in the order of thousands of species.
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Prosser Jl: Molecular marker systems for detection of genetically engineered micro-organisms in the environment. Microbiology 1994, 140:5-17. This is an excellent review of marker/reporter genes for use in the environment.
of microbial nucleic acids, detection of microbial nucleic acid sequences, identification and classification of microbes using DNA and RNA sequences, detection of gene transfer in the environment, tracking of specific microbes in the environment, and designing field and microcosm experiments with GEMs.
11.
Flemming CA, Leung KT, Lee H, Trevors JT, Greet C: Survival of a lux-lac marked biosurfactant-producing Pseudomonas aeruginosa UG2 strain in soil: monitored by non-selective plating and PCR techniques. App/Environ Micmbiol 1994, 60:1606-1613.
22.
Saano A, Tas E, Pippola S, Lindstrom K, Van Elsas JD: Extraction and analysis of microbial DNA from soil. In Nucleic Acids in the Environment: Methods and Appfications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vedag; 1995:49-67.
23.
12.
Van Dyke MI, Lee H, Trevors JT: Survival and polymerase chain reaction detection of luxAB-marked Alcaligenes eutrophus H850 in PCB-contaminated soil and sediment in the presence of rhamnolipid biosurfactants. J Chem Technol Biotechno/1996, 65:115-122.
Bailey MJ: Extraction of DNA from the phyllosphere. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors iT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:89-109.
24.
NesmeX, Picard C, Simonet P: Specific DNA sequences for detection of soil bacteria. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vedag; 1995:112-139.
25.
Pepper IL, Pillai SD: PCR amplification of DNA from root nodules. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:141-151.
26.
Paul JH, Pichard SL: Extraction of DNA end RNA from aquatic environments. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors iT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:154-1 77.
2?.
Bej AK: PCR amplification of DNA recovered from the aquatic environment. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vertag; 1995:179-218.
28.
Rochelle PA, Will JAK, Fry JC, Jenkins GJS, Parkes RJ, Turley CM, Weightman AJ: Extraction and amplification of 16S rRNA genes from deep marine sediments and seawater to assess bacterial community diversity. In Nucleic Acids in the Environment: Methods and Appfications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Vertag; 1995:119-239.
29.
Smalla K, Van Elsas JD: Application of the PCR for detection of antibiotic resistance genes in environmental samples. In Nucleic Acids in the Environment: Methods and Applications. Edited by Trevors JT, Van Elsas JD. Heidelberg: Springer-Verlag; 1995:242-256.
30.
Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science 1994, 263:802-805. The description of GFP as a marker reporter gene for use in biological research. This is an excellent paper that has stimulated a considerable amount of research on the use of GFP as a marker gene.
Pepper IL, Pillai SD: Detection of specific DNA sequences in environmental samples via polymerase chain reaction. In Methods of Soil Analysis Part 2: Microbiological and Biochemical Properties. Madison: Soil Science Society of America; 1994:707-726.
31.
Holben WE: Isolation and purification of bacterial DNA from soil. In Methods of Soil Analysis Part 2: Microbiological and Biochemical Properties. Madison: Soil Science Society of America; 1994:727-751.
21. •
32.
Ryder M: Key issues in the deliberate release of geneticallymanipulated bacteria. FEMS Microbiol Ecol 1994, 15:139-146.
33.
TrevorsJ: Molecular evolution in bacteria. Antonie Van Leeuwenhoek 1995, 67:315-324.
10.
13.
Leung K, Cassidy M, Holmes, Lee H, Trevors JT: Survival of carrageenan-ensapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr in a forest soil: monitored by polymerase chain reaction and viable plating. FEMS Microbiol Eco/ 1995,16:71-82.
14. •
Lorenz MG, Wackernagel W: Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev 1994, 58:563-602. A review on natural genetic transformation in the environment as a mechanism of gene transfer or gene flow. The authors provide an excellent description of the fundamentals of natural genetic transformation and the bacterial species involved.
15.
TrevorsJT: DNA in soil: adsorption, genetic transformation and molecular evolution. Antonie van Leeuwenhoek 1996, in press.
16.
Stewart G J, Garko KA: The effect of divalent cations on the frequency of natural transformation in Pseudomonas stutzeri strain Zobell in marine sediment. Microb Releases 1994, 2:201-207.
1 7.
Garko KA, Stewart GJ: The effect of divalent cations on the binding of divalent cations to marine sediment. Microb Releases 1994, 2:191-199.
18.
Ogram AV, Mathot ML, Harsh JB, Boyle J, Pettigrew CA Jr: Effects of DNA polymer length on its adsorption to soils. Appl Environ Microbiol 1994, 60:393-396.
19.
Cebolla A, Vazquez ME, Palomares AJ: Expression vectors for the use of eukaryotic luciferases with different colors of luminescence. App/ Environ Microbio/1995, 61:660-668.
20. •-
AkkermansADL, Van Elsas JD, De Bruijn FJ (Eds): Molecular Microbial Ecology Manual. Dordrecht: Kluwer Academic Publishers; 1995. This comprehensive manual contains molecular protocols relevant in microbial ecology research. The manual is divided into seven sections: isolation