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MOLECULAR-BASED APPROACHES TO SOIL MICROBIOLOGY

Fluorescent In Situ Hybridization and Micro-autoradiography Applied to Ecophysiology in Soil

^a U.S. Environmental Protection Agency, National Risk Management Research Lab., 26 W. Martin Luther King Dr., MS 421, Cincinnati, OH 45268
^b USDA-ARS, National Soil Tilth Lab., 2150 Pammel Dr., Ames, IA 50011
^c Dep. of Civil, Construction, and Environ. Eng., 486 Town Engineering Bldg., Iowa State Univ., Ames, IA 50011

^* Corresponding author (rogers.shane{at}epa.gov).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION METHODOLOGIES APPLICATION TO SOILS AND... SUMMARY REFERENCES

 
Soil microbial communities perform many important processes, including nutrient cycling, plant–microorganism interactions, and degradation of xenobiotics. The study of microbial communities, however, has been limited by cultural methods, which may greatly underestimate diversity. The advent of nucleic acids technologies allows microbial communities to be quantified and classified without the limitations of cultivation. Fluorescent in situ hybridization (FISH) and other tools of molecular ecology are now being used to investigate community structure and diversity of soils, aquifers, and other natural habitats. Based on these studies, soil microbial communities are diverse and appear to respond to anthropogenic inputs, such as fertilizer, manure, and pollutants, as well as the more well-known constraints imposed by temperature and moisture. Yet most nucleic-acids-based technologies are unable to directly link phylogeny with processes in a manner similar to cultivation-based approaches, restricting the conclusions that can be drawn from the large data sets they generate. Recently, the combination of FISH with microautoradiography (FISH-MAR) allows cells active in processes to be quantified and simultaneously classified phylogenetically. In this review, we discuss how FISH-MAR can be used to quantify the specific microbial phylotype(s) responsible for a microbially catalyzed process. Examples of the use of FISH and FISH-MAR in soils and sediments are described. The capabilities and limitations of these techniques for linking microbial community structure and function are discussed.

Abbreviations: CLSM, confocal laser scanning microscopy • FISH, fluorescent in situ hybridization • MAR, microautoradiography • PCR, polymerase chain reaction.

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION METHODOLOGIES APPLICATION TO SOILS AND... SUMMARY REFERENCES

 
Much of our understanding of microbial ecology has stemmed from the study of microorganisms cultivated from diverse natural environments. Accurate descriptions of the complex metabolic interactions within these diverse systems based on data obtained using only cultivated microorganisms has proven problematic, however. This may be rooted in the fact that only a small fraction of natural microbial communities (0.1–10%) have been successfully cultivated (Winogradsky, 1949; Ward et al., 1992; Amann et al., 1995; Head et al., 1998; Janssen, 2006). Advances in molecular microbial ecology within the last 20 yr have provided means by which the uncultivated fraction of natural microbial communities can be identified. Whole groups of uncultivated and uncharacterized microorganisms, known only from their nucleic acid sequences, have emerged as significant members in many environments. The relationship between in situ physiology and microbial community remains ambiguous in terrestrial ecosystems.

Fluorescent in situ hybridization (FISH) is one of many nucleic acids techniques useful for studying microorganisms in their natural environments. The technique, first applied by DeLong et al. (1989), uses the ability of dye-labeled oligonucleotide probes to selectively bind to the bacterial ribosome. The dyed cells can then be counted with epifluorescence microscopy, confocal laser scanning microscopy (CLSM), or flow cytometry. Application of these procedures with multiple oligonucleotide probes can provide a general picture of microbial diversity. Alternatively, the use of highly specific probes can provide information on one or several species. The use of 16S-rRNA FISH and related approaches have been reviewed previously (Amann et al., 1995; Moter and Göbel, 2000). Probes are available in hierarchical sets from the domain level (Bacteria and Archaea) to the species level based on the 16S rDNA database.

Microautoradiography (MAR) is a process by which radioactive materials taken up by cells are visualized by exposure of a thin film of photographic emulsion on a microscope slide. The exposed emulsion creates a pattern of silver grains surrounding the cells that can be seen clearly by transmission light microscopy. Recently, two independent research groups introduced the coupling of FISH with microautoradiography (FISH-MAR) facilitating the phylogenetic identification of substrate-active cells within complex microbial communities (Lee et al., 1999; Ouverney and Fuhrman, 1999). This is a significant advancement over earlier MAR techniques based on morphology, general staining, or antibody staining because it allows investigators to answer questions regarding the metabolic state of microbial communities without a priori knowledge of the microbial community of interest, or when morphology and phylogeny may not reflect well cellular physiology (Fliermans and Schmidt, 1975; Meyer-Reil, 1978; Fuhrman and Azam, 1982; Tabor and Neihof, 1982, 1984; Andreasen and Nielsen, 1997). The FISH-MAR technique has proven useful for the study of diverse environments including marine and fresh waters and sediments, hot-spring microbial mats, and contaminated aquifer sediments (Lee et al., 1999; Ouverney and Fuhrman, 1999; Otte et al., 1999; Gray et al., 2000; Nübel et al., 2002; Ito et al., 2002; Glaeser and Overmann, 2003; Malmstrom et al., 2004; Villa et al., 2004; Rogers, 2004). This review examines these techniques and describes their application to soils and sediments.

	METHODOLOGIES

TOP NOTES ABSTRACT INTRODUCTION METHODOLOGIES APPLICATION TO SOILS AND... SUMMARY REFERENCES

 
The basic methodology for FISH-MAR in soils and sediments can be divided into five steps including incubation with radioactive substrates, fixation and extraction, FISH, MAR, and microscopy (visualization). Variations in these methodologies are discussed to facilitate effective adaptation of these techniques.

Incubation with Radioisotopic Compounds
The FISH-MAR technique can be used to identify the general metabolic activity of a microbial community (Nielsen et al., 2003a), to study the in situ physiology of a defined (probe-specific) groups of microorganisms (Gray et al., 2000), or to identify and quantify microorganisms responsible for a physiological process of interest in the environment (Nielsen and Nielsen, 2002; Gieseke et al., 2005). Incubation conditions and radioactive substrates are chosen depending on the objective of the study. For instance, substrates such as amino acids, glucose, and acetate have been used to identify overall metabolic activity, and may be more sensitive than alternative methods to detect viability such as reduction of the redox dye 5-cyano, 2,3-tolyl-tetrazolium chloride (Vollertsen et al., 2001; Nielsen et al., 2003b). Leveraging the use of these or more specific substrates with controlled incubation conditions has also proven useful for identifying the metabolic activity of specific functional groups such as autotrophic, mixotrophic, and heterotrophic bacteria under aerobic, denitrifying, Fe-reducing, and sulfate-reducing conditions in complex communities (Ito et al., 2002; Nielsen and Nielsen, 2002; Nielsen et al., 2002; Thomsen et al., 2004; Güven et al., 2005). Microbial processes have also been studied with more specific substrates such as radioisotopic orthophosphate for quantifying phosphate-accumulating organisms (Kong et al., 2002, 2004; Lee et al., 2003).

The incubation period is a critical factor in study design. Where the intrinsic microbial community structure is of interest, incubation periods must be minimized to reduce potential divergence caused by the selective pressures of incubations. Enough time must be given, however, to allow sufficient absorption or cellular incorporation of the radioisotope (but not necessarily cell doubling) to detect cells using microautoradiography. Therefore, the appropriate incubation conditions can vary depending on the metabolic activity of the community of interest and the substrate used, and will rarely be known a priori. For more active microbial communities such as those derived from wastewater treatment plants or natural surface waters, short incubation times, generally ranging from 2 to 4 h, have been reported (Andreasen and Nielsen, 1997; Lee et al., 1999; Kindaichi et al., 2004; Ginige et al., 2004). In less active microbial communities or those consuming more challenging substrates such as xenobiotic compounds, longer incubation periods of up to 2 d have been required to achieve visible autoradiograms (Yang et al., 2003; Rogers, 2004). Due to these uncertainties, each new study should investigate the effects of incubation time on both the autoradiographic response and divergence of microbial communities from their initial structure.

Cell Fixation and Extraction
Cell fixation stabilizes and permeabilizes microbial cells for later introduction of oligonucleotide probes during the FISH step. Fixation can be performed following extraction of cells from the soil or directly in the soil matrix. Solutions of paraformaldehyde or formaldehyde with dehydrating ethanol washes are common fixatives. Modifications of these procedures to optimize staining of Gram-positive microorganisms include ethanol and lysozyme pretreatments to increase cell permeability and reduce potential cross-linking of paraformaldehyde that can lower cell-wall permeability, and are described by Moter and Göbel (2000). It appears unlikely that a single fixative will achieve optimal results for all cell types. For mixed microbial communities, however, Hugenholtz et al. (2001) suggested that paraformaldehyde fixation will yield satisfactory results.

In some instances, the association of microbial cells with the soil matrix is of interest. In these cases, differential extraction techniques of increasing rigor can be used to identify less tightly bound cell types apart from those that are closely associated with soil particles (Ranjard et al., 2000). Further, using CLSM and fluorescent dyes, it is possible to visualize spatial distributions of microorganisms directly in association with soil aggregates (Li et al., 2004). Although FISH has not yet been applied in such a fashion, it has been shown to be useful for visualizing bacterial cells bound to soil particles (Christensen et al., 1999), and therefore should be amenable to similar studies. Alternatively, stabilizing agents such as agarose or epoxy resins may aid in the study of microbial structure in relation to soil aggregates, but have yet to be explored in detail with FISH methods (see discussion below). Advances in probe technologies, fluorescence microscopy, and digital imaging should focus on reducing background fluorescence of soils and related organic materials, and increasing microscopic resolution.

If the intact structure of the microbial community is of less interest than identifying and quantifying the community composition, then extraction of cells from the soil matrix can increase the sensitivity of the FISH technique considerably. Extraction of microorganisms from the soil matrix includes reduction in background fluorescence, allowing the detection of microorganisms that would otherwise be blocked from view by soil particles, and the concentration of microorganisms, increasing the probability of detecting groups of microorganisms present in smaller populations. Therefore, microbial cell extraction can be an important aspect in the application of FISH to soil and sediment samples. Ideally, methods should maximize recovery of intact cells, remove interfering mineral or organic substances that can affect background fluorescence, and result in a community structure equivalent to that of the parent soil or sediment.

Cell extraction methods were originally developed for direct microscopic cell counting. Known volumes of diluted soil or water were spread directly on slides (the soil smear) and stained (Jones and Mollison, 1948) or passed through filters (Hobbie et al., 1977). Further modifications (extractants, centrifugation steps) were tested in the course of ecological studies of Rhizobium and other bacteria using fluorescent antibodies (Bottomley, 1994). A variety of extraction solutions have been commonly included phosphate buffer, sodium pyrophosphate, cation exchange resins, and detergents such as Tween (polysorbate). These solutions, when combined with mechanical agitation or sonication, act to disperse soil microaggregates and release the bacteria. Lindahl and Bakken (1995) found that shaking and sonication resulted in greater cell recoveries than blenders or other harsh mechanical treatments and recommended extraction solutions with a pH of 8 to 9. More recently, a method has been proposed for marine sediments that used 10% methanol as an extractant (Lunau et al., 2005). The methanol extractant was superior to pyrophosphate and Tween 80 based extractions in marine sediments. We are not aware of the use of this method in soils. The soil smear technique has been used in a number of recent studies (Kobabe et al., 2004; Nogales et al., 2001; Zarda et al., 1997). The advantages of the soil smear technique are simplicity and ease.

Extraction procedures followed by density gradient centrifugation have also been widely used. These methods use soil dispersion followed by low-speed centrifugation or a settling period to remove sand and silt particles. Nycodenz gradients, developed by Lindahl and Bakken (1995) appear to be effective for soils and sediments. Extracted cells can be applied directly to a Nycodenz gradient (Axis-Shield PoC AS, Oslo, Norway) and concentrated by high-speed centrifugation, effectively removing clays and aggregating bacteria near the Nycodenz–water interface. Centrifugation-based methods have the potential to concentrate cells and remove soil and organic materials that contribute to background autofluorescence, which should allow easier detection of smaller populations. Polyvinylpyrrolidione can be added to extraction solutions to remove humic acids, which contribute to background autofluorescence. Treatment of extracts with 0.01% (w/v) of toluidine blue has been reported to reduce background fluorescence of soil particles (Weber et al., 2001).

Fluorescent In Situ Hybridization
The FISH technique relies on specific binding of a constructed oligonucleotide probe complimentary to the rRNA of an organism of interest. Construction of an oligonucleotide probe, therefore, requires at least partial knowledge of the rRNA sequence of the target cell. The most common target sequences are within the 16S subunit of the bacterial ribosome, but other ribosome subunit sequences have also been used.

Oligonucleotide probes typically consist of 18 to 30 nucleotide bases conjugated to a fluorescent marker on the 5' end that allows detection of probes bound to cellular rRNA by fluorescence microscopy. By conjugating probes with different fluorochromes to selected microbial targets, several target organisms can be visualized at the same time. Probe fluorochromes should be chosen to yield the largest degree of contrast to potential autofluorescence from soil components.

Probes suitable for use in FISH have been developed at a variety of taxonomic levels (Table 1). At the domain level, the EUB338 probes (I, II, and III) target the Bacteria, while the Archaea are stained by the ARCH915 probe and the Eukaryota by the EUK516 probe. More specific probe sets for lower taxonomic levels are described in published literature, or can be constructed using consensus rDNA data published on large public access databases (e.g., the Ribosomal Database Project II [Cole et al., 2005]). Detailed protocols for the design and evaluation of 16S-rRNA-targeted oligonucleotide probes for use in environmental samples are available (Hugenholtz et al., 2001). Alternatively, a useful public access database for selection of FISH probes at various taxonomic levels that have been cataloged from published literature is probeBase (http://www.microbial-ecology.net/probebase/default.asp [verified 16 Dec. 2006]; Loy et al., 2003). As new 16S rDNA sequences and oligonucleotide probes are continually identified and added to public access databases, the consensus sequences need to be periodically checked.

View this table: [in this window] [in a new window]   Table 1. Examples of group-specific oligonucleotide probes relevant to soil microbial communities.

Methods used in FISH-MAR for oligonucleotide staining are generally similar to those described above. The most important consideration is performing the FISH procedures before MAR so that silver granules deposited adjacent to radioactive cells during MAR are not dislodged during the hybridization steps (Ouverney and Fuhrman, 1999; Cottrell and Kirchman, 2000). Of further concern is the ability to visualize cells that may be masked behind dense silver grain formations. To overcome this limitation, cells can be fixed to gelatin-, poly-L-lysine-, or 3-aminopropyltriethoxysilane-coated microscope cover slides by drop mounting or transferring from polycarbonate filters using a sandwich technique before FISH and MAR (Lee et al., 1999; Cottrell and Kirchman, 2000; Nielsen et al., 2003a). In this way, cells are located between the microscope objective lens and the underlying silver grain clusters, making them visible with fluorescence microscopy when mounted onto microscope slides following MAR. Alternatively, FISH can be performed on polycarbonate filters, which are then placed cell-side down onto autoradiographic-emulsion-coated slides. The filter is then peeled away following slide development (Malmstrom et al., 2004). Greater than 80% retention of cells to slides can be achieved using these techniques (Malmstrom et al., 2004). Cells should be extracted from soils before FISH-MAR because soil particles can interfere with resolving silver grain density images. Soils may also bind hydrophobic radioisotopic substrates, yielding false-positive results or severe autoradiographic background signals.

A common difficulty for the application of FISH to soils and sediments is a low signal-to-noise ratio. This may be caused by excessive background fluorescence from soils, sediments, or plant materials that are co-located or co-extracted with microbial cells or low activity of cells (low numbers of ribosomes per cell) that affects the total number of fluorochromes (fluorescence intensity) per cell. Aside from differential staining techniques and cell extraction methods described above to reduce background fluorescence, several strategies have been proposed that increase the probe specificity or fluorescence signals of FISH probes to cope with these issues. The simplest solutions include the use of newer marker dyes including the Cy dyes (especially Cy3), Alexa dyes, FAM, and TAM. These markers have advantages over older fluorescein (FITC, FLUOS) and rhodamine (TRITC) derivatives of being brighter and having less tendency to fade during the microscopic examination. Another simple solution is attaching a fluorochrome at both the 3' and 5' ends of the oligonucleotide probe to effectively double the fluorescence signal.

There are several other options for increasing the fluorescence of stained cells in soil- or sediment-derived samples. Pernthaler et al. (2002) compared polyribonucleotide probes to oligonucleotide probes in enumeration of Bacteria and Archaea. Polyribonucleotide probes are long (>100 bases) rRNA transcripts of 16S rDNA that contain numerous fluorochrome-labeled bases. The polyprobes have the advantage of greater fluorescence, but require careful fixation and hybridization to allow these larger molecules to enter the cells. Another approach, catalyzed reporter deposition (CARD-FISH), uses tyramide signal amplification (TSA) following hybridization of horseradish peroxidase-linked oligonucleotide probes to 16S rRNA in target cells. The peroxidase acts on fluorochrome tyramides to leave deposits of fluorochrome adjacent to the oligonucleotide inside the target cell (Bobrow et al., 1989; Pernthaler et al., 2002). The advantage of this technique is increased brightness, which may contribute to an increased ability to detect populations. The fixation and hybridization procedures may require modification to allow these larger molecules to enter the cells. A unique adaptation of the TSA technique is the use of quantum dots (nanocrystals with a CdSe core and ZnS shell coated with a hydrophilic polymer layer) in lieu of traditional fluorochromes, which results in a much brighter and more photostable stain (Ness et al., 2003; Alivisatos et al., 2005). Although the joint application of CARD-FISH and quantum dots has yet to be reported, there are no apparent reasons why these technologies cannot be combined to improve fluorescence signals in a FISH format. Peptide nucleic acid probes (PNA), which contain a polyamide backbone instead of the sugar-phosphate component of nucleic acids, are another alternative to the conventional oligonucleotide. These probes are reported to be more stable and bind more strongly than oligonucleotides (Lehtola et al., 2005). Finally, PNA molecular beacon probes offer an improved signal-to-noise ratio compared with DNA oligonucleotide probes (Xi et al., 2003). These probes are designed to take advantage of fluorescence resonance energy transfer by folding into a hairpin loop at low temperatures, bringing into close contact the fluorochrome and a quencher molecule, which are covalently linked to the 5' and 3' ends of the oligonucleotide probe. When applied at high temperature and hybridized to target RNA, the probes are unfolded, thus maximizing the distance between the fluorochrome and quencher molecule and resulting in a bright signal when excited at the appropriate wavelength. The inclusion of a hairpin loop in probe design provides an intrinsic competitor for probe binding, increasing probe specificity to complimentary rRNA.

Microautoradiography
Microautoradiography relies on the detection of decaying radioisotopic compounds via exposure of a thin layer of highly sensitive photographic emulsion. Briefly, radioactive cells release beta decay particles into the emulsion during exposure, which reduce the silver ions in silver halide crystals to silver atoms. When developed, additional reduction of silver ions occurs preferentially where atomic silver is already present, resulting in small "clumps" of silver metal (silver grains) adjacent to radioactive cells. These silver grains can be seen clearly by transmission light microscopy.

Microautoradiography should be performed in complete darkness and according to the manufacturer's instructions. To reduce film thickness and the density of silver grains (making microscopic visualization more manageable), however, autoradiography emulsion is typically diluted up to 1:3 with ultrapure water, gelatin, or agarose before use (Ouverney and Fuhrman, 1999; Cottrell and Kirchman, 2000; Teira et al., 2004; Alonso and Pernthaler, 2005). Care must be taken to not thin the film too much, which can result in uneven distribution over bioflocs or smaller cell aggregates (Nielsen et al., 2003a). Quality control procedures for microautoradiography should include: (i) replicates of each sample, (ii) positive and negative control strains for uptake of the radioisotope(s) of interest, (iii) paraformaldehyde-fixed or pasteurized samples incubated with the radioisotope(s) of interest to check for adsorption phenomena, and (iv) samples incubated without added radioactivity to check for chemography.

Exposure times for microautoradiography cannot be predicted a priori, as they can range from a few hours to weeks depending on several factors including, but not limited to: (i) the decay rate of the radioisotope used, (ii) the concentration of substrate used in the incubation, (iii) the label position and specific activity of the radioisotope, (iv) the specific cellular uptake rate, and (v) the age and dilution of the autoradiographic emulsion used. A time-course study can identify the appropriate exposure time to be used. The optimum exposure time maximizes the number of fluorescent cells that can be associated with clusters of silver grains. Clusters of silver grains are identified relative to the density of the background of silver grains (i.e., number per square micrometer), which increase with the time of exposure due to several factors including, but not limited to, the duration of exposure to decaying isotopes, incidental exposure to light, and chemography. Cells with known radioactivity can also be used as quantitative quality control standards for identifying appropriate exposure times or quantifying cell-specific substrate uptake rates (Nielsen et al., 2003a).

Microscopy
The FISH-MAR cover glasses or slides should be mounted with a low fluorescence antifade mountant before microscopic analysis. Microautoradiography-positive (MAR+) cells are counted using transmission light microscopy and probe-stained cells can be enumerated using an epifluorescence microscope or a confocal laser-scanning microscope. Switching between bright-field and fluorescence modes for each microscopic field allows MAR+ probe-defined cell types to be identified. Care must be taken to consistently identify MAR+ cells, which should be clearly distinguishable from potential background silver grain formation. No set rules for identifying silver grain clustering from background silver grain formation exist. Nielsen et al. (2003a), however, examined the density of silver grains within either 1 m of cells consuming ³H-labeled substrates or 5 m of cells consuming ¹⁴C-labeled substrates, and defined clusters of silver grains as those that were 100 times denser than the background at 10 m from either side of MAR+ cells. Their definitions are easily adapted for use in image analysis software to automate identification of silver grain clusters by size exclusion (Rogers, 2004).

Image analysis techniques can also be used as another strategy to improve resolution and discrimination between cells and background fluorescence in FISH. These methods use charge-coupled device (CCD) cameras to capture images from the fluorescence microscope. Commercial or free software can then be used to recognize and brighten cells present on the digital images relative to the background (Schönholzer et al., 2002; Selinummi et al., 2005). The use of computer-controlled microscope stages and autofocus allows full automation of the counting process (Thiel and Blaut, 2005). Figure 1 shows an example of digital image processing for FISH-MAR. Black and white images of the same microscopic field, but from two different band pass filters (Bacteria CY3 and DAPI) are each digitally optimized for brightness and contrast, aligned by the image processing software, and assigned individual colors. These images can be composited with the complementary bright-field or phase contrast image to allow simplified detection of MAR+ cells. The green background of the autoradiogram in the composite image provides higher contrast for detecting red and blue cells.

View larger version (78K): [in this window] [in a new window]   Fig. 1. Digital image processing of fluorescent in situ hybridization (FISH)–microautoradiography (MAR). Separate images for each color channel and the phase contrast counter image can be composited into a single image to simplify detection of cells that consume radioisotopic substrates. Stain DAPI (excitation = 385–400 nm, emission = 450–465 nm); probe EUB338-I, II, III (stain CY3; excitation = 530–560 nm, emission = 573–648 nm).

	APPLICATION TO SOILS AND SEDIMENTS

TOP NOTES ABSTRACT INTRODUCTION METHODOLOGIES APPLICATION TO SOILS AND... SUMMARY REFERENCES

The FISH technique has also been used to investigate the structure of smaller microbial communities or visualize more specific microbial targets, some of which belong to bacterial phylotypes that have not been previously cultivated. For example, Eller et al. (2001) described the development and application of probes specific for two groups of methanotrophs. They used multiple fluorochromes and image overlapping to visualize both groups of methanotrophs simultaneously in soil. Ludwig et al. (1997) extracted and polymerase chain reaction (PCR) amplified rDNA from soil, and then cloned several of the PCR products into Escherichia coli. Comparison of these cloned sequences to databases of other rDNA sequences indicated that 50 were representative of a bacterial group containing Holophaga, Geothrix, and Acidobacterium. Specific oligonucleotide probes were constructed, and cells from soil extracts were visualized by FISH.

A unique application of the FISH technique is to examine the spatial relationships of different groups of microorganisms and their comparative abundance in relatively undisturbed samples. Both FISH and CLSM have been used to examine rhizosphere populations of various microorganisms (Assmus et al., 1995; Gilbert et al., 1998; Watt et al., 2006). Stabilization of the physical structure of the sample may be necessary. Macnaughton et al. (1996) used agarose as a stabilizing agent after fixation with paraformaldehyde for FISH analysis of rhizosphere populations, allowing more precise visualization of the distribution of bacteria on plant roots. Epoxy resins have also been used to structurally stabilize samples before examination with FISH (Moter et al.,1998). The use of FISH with resin-embedded soil samples has not yet been systematically examined, but Li et al. (2004) have reviewed many aspects of preparing soil thin sections and staining procedures. Nunan et al. (2001, 2002) prepared epoxy resin thin sections of soil prestained with calcafluor white instead of FISH to explore the spatial variation in bacterial populations in relation to soil aggregate and pore structure. They noted differences between topsoils and subsurface soils in bacterial aggregation related to pore structure. Alternatively, differential cell extraction procedures have also been shown effective to examine microbial community structure in parts of soil microaggregates (see above).

A potential limitation for the use of FISH in environmental matrices, including soil, is that Eubacteria and Archaea probe-detected cells are typically one- to two-thirds that of total cell counts obtained by the commonly used counterstains, acridine orange, SYBR Green, or DAPI, which bind nonspecifically to cellular DNA (Bouvier and del Giorgio, 2003; Eller and Frenzel, 2001; Kobabe et al., 2004; Nogales et al., 2001; Stein et al., 2005; Zarda et al.,1997, 1998; Chatzinotas et al., 1998). Bouvier and del Giorgio (2003) attributed the difference between EUB338- and DAPI-stained cells in aquatic systems to low-intensity fluorochromes, inefficient fixation and hybridization procedures, and low cellular ribosome counts. These factors operate independently or in combination to produce poorly stained cells and undercounted populations, which in turn contribute to the discrepancy between DAPI and EUB338 cell counts in soils. Strategies for overcoming some of these problems are discussed above. Natural environments are also likely to harbor substantial populations detectable by DAPI, but not by EUB338 probes, such as Eukaryotes and Planctomycetes (Neef et al., 1998). Of the group-specific probes presently available for FISH, the within-group coverage ranges from 34 to 95%, potentially resulting in many false negative results. Better probe designs could alleviate some of these issues, but soils may still harbor several uncultured and unsequenced bacteria that are detected by DAPI but not by the EUB338 probe set. Finally, Zweifel and Hagström (1995) and Luna et al. (2002) showed evidence that up to 70% of DAPI-stained cells could be dead or inactive, potentially as a result of DAPI binding to sites created by paraformaldehyde fixation. A more vigorous destaining step may improve discrimination of viable and nonviable cells (Zweifel and Hagström, 1995).

Fluorescent In Situ Hybridization–Microautoradiography
The FISH-MAR technique is emerging as a powerful tool for environmental microbiologists, enabling identification of microorganisms catalyzing specific processes in various environments and the study of the physiology of defined groups of microbes in situ. For instance, FISH-MAR has proven useful for improving cultivation strategies for bacterioplankton from lake waters and studying the in situ distribution and substrate uptake patterns of Chloroflexus spp. in hot-spring microbial mats (Vollertsen et al., 2001; Nübel et al., 2002; Bruns et al., 2003). Although broad in potential, the overwhelming majority of reported applications of FISH-MAR to date have focused on nutrient removal and process control in wastewater treatment and, to a lesser degree, the characterization of natural microbial communities in marine and estuary environments. Many of these applications have been tabulated in previous reviews (Gray and Head, 2001; Wagner and Loy, 2002; Wagner et al., 2006). The paucity of reported applications of FISH-MAR to soils and sediments is perhaps related to the difficulties associated with application of FISH to these matrices, as described above. Using appropriate adaptations to FISH protocols, however, as discussed above, similar objectives could be addressed in regard to soil microbial communities.

For instance, the biogeochemical cycling of N is an important process in soils and aquatic ecosystems affecting soil fertility, plant growth, and water quality. Nitrogen removal is also an important process in wastewater treatment for protection of water quality. Nutrient removal during wastewater treatment relies on nitrification of NH₄⁺ and subsequent denitrification before discharge. Since establishment and maintenance of nitrification in wastewater treatment can be a lengthy and difficult process, there is intense interest in understanding the ecophysiology of nitrifying bacteria that may lead to more stable system performance. The FISH-MAR technique has been used extensively to study the ecophysiology of nitrifying bacteria in wastewater treatment systems, and to identify dominant groups of denitrifying bacteria in mixed microbial communities of bioreactors (Daims et al., 2001; Kindaichi et al., 2004; Gieseke et al., 2005; Ginige et al., 2004; Okabe et al., 2005). Adapting the methodologies of these researchers to study plant–microbe interactions in the root zone or other soil systems should be rapid, and has long been a desirable capability. In fact, before the discovery of FISH techniques, Fliermans and Schmidt (1975) were able to study the ecophysiology of Nitrobacter agilis and N. winogradskyi in soils from Mammoth Cave using fluorescent antibody staining combined with MAR.

Another application of FISH-MAR is the identification microorganisms catalyzing the biodegradation of xenobiotic organic contaminants in polluted soils and quantification of their intrinsic activity. Prediction of biodegradation and transformation processes in soils, subsoils, and aquifer materials is limited by our knowledge of the in situ populations that are active in the biodegradation process, and the interaction of these communities with the environmental conditions. A variety of 16S rDNA analyses indicate that microbial communities in aquifer sediments are diverse and can be dynamic in space and time. Complex food webs may result from the introduction of large, localized C sources into soils or aquifers through pollutant spills or leaks (Carman et al., 1995; Ghiorse et al., 1995; Langworthy et al., 1998).

In a recent work, we applied FISH and MAR to characterize the microbial community of a coal-tar-impacted aquifer in which polycyclic aromatic hydrocarbons (PAHs) were the primary pollutants (Rogers, 2004). In this aquifer, the microbial community was three orders of magnitude greater in population in the coal-tar source region than in nearby unaffected sediments. Spatial heterogeneity in the intrinsic microbial community structure was observed. Actinobacteria, Gammaproteobacteria, Bacteroidetes, and Betaproteobacteria dominated the aerobic (>1 mg/L dissolved O₂) and NO₃–reducing in situ microbial community. Sulfate-reducing bacteria (SRB385, Table 1) were dominant in sediments associated with sulfate reduction. Using FISH-MAR, the uptake of [UL-¹⁴C]naphthalene and [9-¹⁴C]phenanthrene under aerobic conditions were associated with members of the Betaproteobacteria, Gammaproteobacteria, and Actinobacteria (see Fig. 2 ). The number of bacteria taking in naphthalene ranged from 2.4 to 4.5% of the total microbial community (1.0 x 10⁷ to 2.2 x 10⁸ naphthalene-degrading bacteria/kg sediment), and included all three taxa regardless of the sediments investigated. The number of bacteria taking in phenanthrene ranged from 0.8 to 1.0% of the total microbial community (8.0 x 10⁶ to 4.0 x 10⁷ phenanthrene-degrading bacteria/kg sediment), but exhibited differences in the uptake patterns by the Betaproteobacteria and Gammaproteobacteria depending on the aquifer sediments investigated. The combination of FISH and MAR showed that only a small fraction of the intrinsic microbial community could be directly related to the uptake of naphthalene and phenanthrene. This is contrary to current models of biodegradation, which assume a single physiology and constant activity for PAH-degrading bacteria. Direct evidence of intrinsic degradation of PAH pollutants such as that provided by FISH-MAR may be necessary to construct better predictive models of intrinsic biodegradation at complex PAH-contaminated sites (Smets and Pritchard, 2003).

View larger version (63K): [in this window] [in a new window]   Fig. 2. Autoradiogram (left side) and corresponding fluorescence image (right side) following 10-d autoradiographic film exposure to cells from 36-h aerobic incubations of polycyclic aromatic hydrocarbon contaminated aquifer sediments with [9-14C]phenanthrene. Silver grain clusters surrounding bacterial cells indicate active cellular incorporation of [9-14C]phenanthrene (A and B), which can be differentiated from bacteria that do not metabolize [9-14C]phenanthrene (C and D). Pink cells indicate Bacteria [EUB338(I, II, III) and blue cells are DAPI stained. Bar in autoradiogram = 10 µm. MAR = microautoradiography.

Environmental samples with low microbial activities may be problematic for FISH-MAR because they may contain cells of insufficient 16S rRNA to be detectable with FISH or they may lack sufficient metabolic activity to elicit an autoradiographic response. To reduce false negatives, extended incubation times may be required to obtain visible cells and MAR density images; however, this may lead to divergence from the intrinsic microbial community structure and activity. Furthermore, uptake of radioactive metabolites and cellular byproducts of growth on radioactive substrates by organisms that are not primary consumers of the substrate of interest (cross-feeding) will increase with time. For instance, heterotrophic bacteria may take in ¹⁴CO₂ produced during degradation of parent compounds for carboxylation reactions, eventually leading to false positives (Hesselsoe et al., 2005). Therefore, care must be taken to fully characterize the microbial communities before and following FISH-MAR incubations and to use appropriate controls, including negative controls, to discriminate between the true uptake of a parent isotope and experimental artifacts of the MAR procedure.

Aside from these limitations, technical advancements in FISH and MAR methodology are expanding the utility of this technique for both engineered and natural systems. For instance, the full-cycle 16S rRNA approach may be cumbersome and can underemphasize the importance of certain groups in the uptake of specific substrates in situ. Some researchers have been able to couple micromanipulation of intrinsic microbial communities to isolate and identify specific organisms of interest based on FISH in environmental samples (Thomsen et al., 2004), which could make the full-cycle approach less cumbersome. This technique has yet to be adapted, however, to the isolation and identification of MAR+ cells hybridized with group-specific probes. Finally, improvements in quantitative FISH-MAR methodology have been proposed that may offer the ability to identify in situ kinetic coefficients (Nielsen et al., 2003a). Uptake of labeled substrates was quantified by counting cell-associated silver grains after MAR and comparing with internal control standards. Measurements of uptake at different substrate concentrations allowed estimation of best-fit half saturation constants and maximum uptake rates. Addition of a large pool of radioactive substrate at various doses to yield visible silver grain density images for microautoradiography will result, however, in a range of substrate concentrations greater than that of the original environmental sample. Use of the best-fit kinetic constants will implicitly require extrapolation of the fitted model below the range of substrate concentrations used to derive the kinetic coefficients. Interpretation of best-fit kinetic coefficients derived from quantitative FISH-MAR when applied in soils or aquifer sediments may be further complicated by processes such as diffusion or sorption that may affect uptake of radioisotopic substrates.

Complementary and Alternative Techniques
Several techniques can provide alternative or complementary information on the microbial community structure of soils. For instance, Buckley and Schmidt (2003) extracted rDNA followed by quantitative dot-blot hybridization with ³²P-labeled oligonucleotide probes to obtain information that was qualitatively similar to information that could have been obtained by a FISH procedure. Nogales et al. (2001) compared community structure assessed through 16S rDNA and rRNA sequence libraries and FISH and reported qualitative similarities between the techniques. Fierer et al. (2005) applied taxon-specific SYBR Green quantitative polymerase chain reaction (qPCR) assays to rapidly assess the relative abundance of bacteria and fungi in three soils; the results of which are very similar to those for which FISH was used for identifying microbial community structure reported above. Clearly, a variety of rDNA based techniques (qPCR, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, and ribosomal intergenic spacer analysis) have been applied to soil, but recovery of DNA, differences in copy numbers of DNA between bacterial cell types, and subsequent PCR amplification steps introduce potential bias. Construction of sequence libraries can offer more detailed views of community structure and diversity (e.g., Ranjard et al., 2000; Zhou et al., 2004) than those obtained with a limited set of FISH probes, but these efforts may require a greater investment of resources.

The FISH-MAR technique provides information on both community structure and function. At present, the number of microbial cell types that can be detected at once is effectively limited by the number of fluorochromes that can be simultaneously resolved using CLSM. This may result in tedious repetition, especially when attempting to identify microorganisms catalyzing specific processes in natural environments. Advanced beta imaging technologies allow the visualization of radioactivity with a spatial resolution as low as 20 µm. Although this resolution is not fine enough to replace MAR for co-locating FISH-detected cells in a whole-cell format, it does allow for construction of isotope arrays that can be used in a PCR-independent manner to rapidly screen microbial communities of various habitats to identify microorganisms consuming a specific substrate of interest (Adamczyk et al., 2003). Briefly, following incubation with radioisotopic substrates similar to FISH-MAR, whole-community rRNA is extracted, fragmented, conjugated to a fluorescent dye, and hybridized to an rRNA-targeted microarray. A fluorescence scanner and beta imager are used to quantify probe signal and radioactivity (Wagner et al., 2006). The effectiveness of the isotope array approach will depend on the availability of suitable rRNA-targeted oligonucleotide microarrays.

Stable isotope techniques comparable to FISH-MAR include stable isotope probing (SIP) and ¹³C-phospholipid fatty acids analysis (¹³C-PLFA). These techniques rely on incorporation of ¹³C-isotopic substrates into nucleic acids or fatty acids, respectively, followed by high-speed density-gradient fractionation of heavy DNA for cloning and sequencing (SIP), or extraction and mass spectrophotometric analysis of isotopic PLFAs (¹³C-PLFA) to identify the active microbial cell types in a variety of environments, including soil (Madsen, 2006; Griffiths et al., 2004; Butler et al., 2003). The considerations pertaining to substrate incorporation into the active microbial biomass described above are similar for ¹³C-PLFA, SIP, and FISH-MAR. Because the extended incubation times required to achieve cell turnover critical to the SIP technique may alter the microbial community profile, FISH-MAR may be useful to validate results obtained with SIP techniques. For example, Ginige et al. (2004) used both FISH-MAR and stable isotope probing to investigate a denitrifying community in a methanol-fed bioreactor. The FISH-MAR results confirmed the identity of the dominant denitrifying microorganisms originally identified with SIP.

Time-of-flight secondary ion mass spectrometry (Cliff et al., 2002) or laser ablation mass spectrometry (Bruneau et al., 2002) in combination with microscopy to visualize cellular incorporation of the stable isotopes from substrates also yielded information on in situ microbial processes. These mass spectrophotometric techniques have been used to study soil microbial communities following dosing with ¹³C (DeRito et al., 2005), and have been combined with FISH to identify methane-consuming Archaea in anoxic marine sediments (Orphan et al., 2001). When coupled to FISH, these techniques are subject to the same limitations in soils discussed above; however, the added capability of visualizing the ecophysiological interactions of intact soil microbial communities with their environment may yield valuable insights not obtainable with MAR or other similar isotope technologies. Aside from the practical advantages of not using radioactive substrates, stable isotopes may also offer several advantages to radioactive compounds such as their use in the field and application to N transformations. The instrumentation necessary for these techniques, however, may at present be cost prohibitive for some users.

	SUMMARY

TOP NOTES ABSTRACT INTRODUCTION METHODOLOGIES APPLICATION TO SOILS AND... SUMMARY REFERENCES

 
Fluorescence in situ hybridization with rRNA-targeted oligonucleotide probes is a well-established technique capable of providing information regarding the in situ phylogeny of a diverse range of microbial communities without cultivation. The strength of this technique over alternative nucleic acids technologies is that it can rapidly and specifically yield the number and spatial distribution of microorganisms in complex matrices. When combined with MAR, FISH can also yield insight into the functional activity of different microbial cell types in situ. Thus, FISH-MAR can be used to simultaneously study the microbial community structure and ecophysiology in a diverse range of habitats, and may also prove useful for improving cultivation strategies for microorganisms in complex matrices that have resisted cultivation to date (Bruns et al., 2003). Most importantly, structure–functions techniques such as FISH-MAR may allow construction of better models of microbial processes in both natural and agricultural systems. These improved models may lead to a better understanding of soil systems.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION METHODOLOGIES APPLICATION TO SOILS AND... SUMMARY REFERENCES

 
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Received for publication March 8, 2006.

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