Techniques For Imaging The Location Of Enzymes

Thin-section techniques are combined with histochemical and imaging techniques to visualize the location of enzymes and their activities. Early papers by Foster et al. (1983) showed peroxidase, succinic dehydrogenase, and acid phos-phatase bound to roots, bacterial cell walls, and organic matter in soil by using either transmission electron microscopy or scanning electron microscopy. In the future, atomic force microscopy, which measures small-scale surface topographical features, will help us to understand enzyme-clay interactions. Confocal microscopy has the potential to reconstruct the detailed three-dimensional distribution of enzyme proteins in soils.

Enzymatic properties of single cells have been screened in the aquatic environment, biofilms, and activated sludge by a new type of fluorogenic compound, ELF-97 (Molecular Probes, Eugene, OR, USA), which is combined with sugar, amino acid, fatty acid, or inorganic compounds such as sulfate or phosphate. This substrate is converted to a water-insoluble, crystalline, fluorescent product at the site of enzymatic hydrolysis, thus reporting the location of active enzymes when viewed by fluorescence microscopy. Kloeke and Geesey (1999) combined this technique with a 16S rRNA oligonucleotide probe specific for the Cytophaga-Flavobacteria group to prove the importance of these microorganisms in liberating inorganic orthophosphate in discrete, bacteria-containing areas of the floc matrix in aerobic activated sludge (Fig. 3.6). Phosphatase activity was primarily localized in the immediate vicinity of the bacterial cells rather than dispersed throughout the floc or associated with rotifers (see Figs. 3.6A and 3.6B). These new substrates have the potential to be used for visualizing the location of enzyme activities in microenvironments in soil.

functional diversity

Microbial diversity reflects the variation in species assemblages within a community. A broader view of functional diversity has advanced our understanding of

FIGURE 3.6 (A) Phase-contrast photomicrograph of activated sludge floc material and associated microorganisms. Arrows indicate protozoa grazing on the floc particles. (B) Epifluorescence photomicrograph of the same field of view as in (A) of whole floc material revealing the areas of intense phosphatase activity (yellow spots where ELF crystals have precipitated, arrow). (C) Epifluorescence photomicrograph of thin section of a floc particle stained red-orange with acridine orange, revealing discrete regions within the floc displaying phosphatase activity (green spots) as a result of incubation in the presence of ELP-P. (D) Epifluorescence photomicrograph image of a homogenized activated sludge filtrate showing floc bacteria from the Cytophaga-Flavobacteria group by using a filter combination that reveals cells that react with CF319a oligonucleotide probe (red). (E) Epifluorescence photomicrograph of the same field of view as in (D) using a filter combination that resolves crystals of ELF (green spots), indicating areas of phosphatase activity. (F) Merged image of (D) and (E), revealing the subpopulation of cells that react positively with the CF319a and also display phosphatase activity (according to Kloeke and Geesey, 1999; pictures are reproduced with the permission of Springer-Verlag).

FIGURE 3.6 (A) Phase-contrast photomicrograph of activated sludge floc material and associated microorganisms. Arrows indicate protozoa grazing on the floc particles. (B) Epifluorescence photomicrograph of the same field of view as in (A) of whole floc material revealing the areas of intense phosphatase activity (yellow spots where ELF crystals have precipitated, arrow). (C) Epifluorescence photomicrograph of thin section of a floc particle stained red-orange with acridine orange, revealing discrete regions within the floc displaying phosphatase activity (green spots) as a result of incubation in the presence of ELP-P. (D) Epifluorescence photomicrograph image of a homogenized activated sludge filtrate showing floc bacteria from the Cytophaga-Flavobacteria group by using a filter combination that reveals cells that react with CF319a oligonucleotide probe (red). (E) Epifluorescence photomicrograph of the same field of view as in (D) using a filter combination that resolves crystals of ELF (green spots), indicating areas of phosphatase activity. (F) Merged image of (D) and (E), revealing the subpopulation of cells that react positively with the CF319a and also display phosphatase activity (according to Kloeke and Geesey, 1999; pictures are reproduced with the permission of Springer-Verlag).

the significance of biodiversity to biochemical cycling. This includes several levels of resolution: (1) the importance of biodiversity to specific biogenic transformations, (2) the complexity and specificity of biotic interactions in soils that regulate biogeo-chemical cycling, and (3) how biodiversity may operate at different hierarchically arranged spatial and temporal scales to influence ecosystem structure and function (Beare et al., 1995). Most methods used for measuring functional diversity consider only the importance of biodiversity of those groups that regulate biochemical cycling. Several approaches enable functional diversity to be measured in situations in which taxonomic information is poor. These include using binary biochemical and physiological descriptors to characterize isolates, evaluating enzymatic capabilities for metabolizing particular substrates, extracting DNA and RNA from the soil, and probing genes that code for functional enzymes. Recent advances in genomic analysis and stable isotope probing are the first steps toward resolving a better linkage between structure and function in microbial communities (see also Chaps. 4, 7, and 8).

Commercially available Biolog bacterial identification system plates or community-level physiological profiles (CLPP) have been used to assess functional diversity of microorganisms, based on utilization patterns of a wide range (up to 128) of individual C sources. The culturable subpart of the microbial community, which exhibits the fast growth rates typical of r-strategists, primarily contributes to CLPP analysis. Preston-Mafham et al. (2002) assessed the pros and cons of its use and point out inherent biases and limitations and possible ways of overcoming certain difficulties. A modification of the SIR method (Degens and Harris, 1997) involves determining patterns of in situ catabolic potential as a measure of functional diversity. These profiles are determined by adding a range of simple C substrates to the soils and measuring short-term respiration response. The use of whole soil in a microplate assay as described by Campbell et al. (2003) can also be used to explore the metabolic capacity of the soil microbial community. An alternative approach was proposed by Kandeler et al. (1996), using the prognostic potential of 16 soil microbial properties, including microbial biomass, soil respiration, N-mineralization, and analyses for 13 soil enzymes involved in cycling of C, N, P, and S. Multivariate statistical analysis is used to calculate the functional diversity from measured soil microbial properties. The latter approach is based on the following assumptions: The composition of the microbial species assemblage (taxonomic diversity) determines the community's potential for enzyme synthesis. The actual rate of enzyme production and the fate of produced enzymes are modified by environmental effects as well as by ecological interactions. The spectrum and amount of active enzymes are responsible for the functional capability of the microbial community irrespective of being active inside or outside the cell. Presence or absence of a certain function, as well as the quantification of the potential of the community to realize this function, has to be considered in ecological studies. This approach may permit evaluation of the status of changed ecosystems (e.g., by soil pollution, soil management, global change) while providing insight into the functional diversity of the soil microbial community of the undisturbed habitat.

Physiological methods are applied to understand the physiology of single cells and soil biological communities, as well as biogeochemical cycling in terrestrial ecosystems. Small-scale studies explain biological reactions in aggregates, in the rhizosphere, or at the soil-litter interface. Combining physiological and molecular methods helps us to understand gene expression, protein synthesis, and enzyme activities at the micro- and nanoscales. Linking these methods can also explain whether the abundance and/or the function of organisms is affected by soil management, environmental change, or soil pollution.

At the field scale, researchers use biochemical and physiological methods to investigate the functional response of soil organisms to the manipulation or preservation of soils. These applications include microbe-plant interactions and controlling plant pathogens, as well as understanding organic matter decomposition and its impact on local and global C and N cycling. Soil biologists investigate the effects of soil management (tillage, fertilizer, pesticides, crop rotation) or disturbance on the function of soil organisms. In many cases, soil microbial biomass and/or soil microbial processes can be early predictors of the effects of soil management on soil quality and can indicate the expected rapidity of these changes. Monitoring of soil microbial properties is also included in environmental studies that test the use of soil microorganisms in bioremediation and composting. Future challenges in functional soil microbiology are to use our present knowledge to scale-up these data to the regional and global scale.

references and suggested reading

Alef, K., and Nannipieri, P. (1995). "Methods in Applied Soil Microbiology and Biochemistry."

Academic Press, San Diego. Anderson, J. P. E., and Domsch, K. H. (1978). A physiological method for the quantitative measurement of microbial biomass in soils. Soil Biol. Biochem. 10, 215-221. Bááth, E., and Anderson, T. H. (2003). Comparison of soil fungal/bacterial ratios in a pH gradient using physiological and PLFA-based techniques. Soil Biol. Biochem. 35, 955-963. Baldocchi, D. D. (2003). Assessing the eddy covariance technique for evaluating carbon dioxide exchange rates of ecosystems: past, present and future. Global Change Biol. 9, 479-492. Beare, M. H., Coleman, D. C., Crossley, D. A., Hendrix, P. F., and Odum, E. P. (1995). A hierarchical approach to evaluating the significance of soil biodiversity to biochemical cycling. Plant Soil 170, 5-22.

Beck, T., Joergensen, R. G., Kandeler, E., Makeschin, F., Nuss, E., Oberholzer, H. R., and Scheu, S. (1997). An inter-laboratory comparison of ten different ways of measuring soil microbial biomass C. Soil Biol. Biochem. 29, 1023-1032. Bloem, J., Bolhuis, P. R., Vininga, R. M., and Wierenga, J. (1995). Microscopic methods to estimate the biomass and activity of soil bacteria and fungi. In "Methods in Soil Microbiology and Biochemistry" (K. Alef and P. Nannipieri, eds.). Academic Press, New York. Boutton, T. W., and Yamasaki, S.-I. (1996). "Mass Spectrometry of Soils." Dekker, New York. Burlage, R. S., Atlas, R., Stahl, D., Geesey, G., and Sayler, G. (1998) "Techniques in Microbial

Ecology." Oxford Univ. Press, New York/Oxford. Burns, R. G. (1982). Enzyme activity in soil: location and possible role in microbial ecology. Soil Biol. Biochem. 14, 423-427.

Burns, R. G., and Dick, R. P. (2002). "Enzymes in the Environment—Activity, Ecology, and

Applications." Dekker, New York. Brookes, P. C., Landman, A., Puden, G., and Jenkinson, D. S. (1985). Chloroform fumigation and the release of soil nitrogen: a rapid direct extraction method to measure microbial biomass nitrogen in soil. Soil Biol. Biochem. 17, 837-842.

Campbell, C. D., Chapman, S. J., Cameron, C. M., Davidson, M. S., and Potts, J. M. (2003). A rapid microtiter plate method to measure carbon dioxide evolved from carbon substrate amendments so as to determine the physiological profiles of soil microbial communities by using whole soil. Appl. Environ. Microbiol. 69, 3593-3599.

Coleman, D. C., and Fry, B., eds. (1991). "Carbon Isotope Techniques." Academic Press, San Diego.

Degens, B. P., and Harris, J. A. (1997). Development of a physiological approach to measuring the catabolic diversity of soil microbial communities. Soil Biol. Biochem. 29, 1509-1320.

Driver, J. D., Holben, W. E., and Rillig, M. C. (2005). Characterization of glomalin as a hyphal wall component of arbuscular mycorrhizal fungi. Soil Biol. Biochem. 37, 101-106.

Eisenbeis, G., Lenz, R., and Heiber, T. (1999). Organic residue decomposition: the minicontainer-system—a multifunctional tool in decomposition studies. Environ. Sci. Pollut. R. 6, 220-224.

Ettema, C. H., and Wardle, D. A. (2002). Spatial soil ecology. Trends Ecol. Evol. 17, 177-183.

Fierer, N., Schimel, J. P., and Holden, P. A. (2003). Variations in microbial community composition through two soil depth profiles. Soil Biol. Biochem. 35, 167-176.

Foster, R. C., Rovira, A. D., and Cock, T. W. (1983). "Ultrastructure of the Root-Soil Interface." Am. Phytopathol. Soc., St. Paul, MN.

Frankland, J. C., Dighton, J., and Boddy, L. (1991). Fungi in soil and forest litter. In "Methods in Microbiology" (R. Grigorova and J. R. Norris, eds.), Vol. 22, pp. 344-404. Academic Press, London.

Fujie, K., Hu, H. K., Tanaka, H., Urano, K., Saitou, K., and Katayama, A. (1998). Analysis of respiratory quinones in soil for characterization of microbiota. Soil Sci. Plant Nutr. 44, 393-404.

Gattinger, A., Gunther, A., Schloter, M., and Munch, J. C. (2003). Characterisation of Archaea by polar lipid analysis. Acta Biotechnol. 23, 21-28.

Gerhardt, P. G., Murray, R. G. E., Wood, W. A., and Krieg, N. R. (1994). "Methods for General and Molecular Bacteriology." Am. Soc. Microbiol., Washington, DC.

Grigorova, R., and Norris, J. R., eds. (1991). "Methods in Microbiology," Vol. 22. Academic Press, London.

Harris, D., Voroney, R. P., and Paul, E. A. (1997). Measurement of microbial biomass N:C by chloroform fumigation-incubation. Can. J. Soil Sci. 77, 507-514.

Herbert, R. A. (1991). Methods for enumerating microorganisms and determining biomass in natural environments. In "Methods in Microbiology" (R. Grigorova and J. R. Norris, eds.), Vol. 22, pp. 1-39. Academic Press, London.

Horwath, W. R., Paul, E. A., Harris, D., Norton, J., Jagger, L., and Horton, K. A. (1996). Defining a realistic control for the chloroform fumigation-incubation method. Can. J. Soil Sci. 76, 459-467.

Hu, H. Y., Lim, B. R., Goto, N., and Fujie, K. (2001). Analytical precision and repeatability of respiratory quinones for quantitative study of microbial community structure in environmental samples. J. Microbiol. Methods 47, 17-24.

Jenkinson, D. S., and Powlson, D. S. (1976). The effects of biocidal treatments on metabolism in soil. V. A method for measuring soil biomass. Soil Biol. Biochem. 8, 209-213.

Joergensen, R. G. (1996). The fumigation-extraction method to estimate soil microbial biomass: calibration of the Kec value. Soil Biol. Biochem. 28, 25-31.

Kammann, C., Grünhage, L., and Jäger, H. J. (2001). A new sampling technique to monitor concentrations of CH4, N2O and CO2 in air at well-defined depths in soils with varied water potential. Eur. J. Soil Sci. 52, 297-303.

Kandeler, E., Kampichler, C., and Horak, O. (1996). Influence of heavy metals on the functional diversity of soil microbial communities. Biol. Fertil. Soils 23, 299-306.

Kandeler, E., Tscherko, D., and Spiegel, H. (1999). Long-term monitoring of microbial biomass, N-mineralization and enzyme activities of a chernozem under different tillage management. Biol. Fertil. Soils 28, 343-351.

Katayama, A., and Fujie, K. (2000). Characterization of soil microbiota with quinone profile. In "Soil Biochemistry" (J. M. Bollag and G. Stotzky, eds.), Vol. 10, pp. 303-347. Dekker, New York.

Kjelleberg, S. (1993). "Starvation in Bacteria." Plenum, New York.

Klee, A. J. (1993). A computer program for the determination of most probable number and its confidence limits. J. Microbial. Methods 18, 91-98.

Klironomos, J. N., Rilling, M. C., and Allen, M. F. (1999). Designing belowground field experiments with the help of semi-variance and power analyses. Appl. Soil Ecol. 12, 227-238.

Kloeke, F. V., and Geesey, G. G. (1999). Localization and identification of populations of phosphatase-active bacterial cells associated with activated sludge flocs. Microbial Ecol. 38, 201-214.

Klose, S. (2003). "Enzyme Mediated Reactions and Microbial Biomass of Agricultural and Fly Ash Influenced Forest Ecosystem." Dresden University of Technology, Dresden. [Habilitation dissertation]

Knowles, R., and Blackburn, T. H., eds. (1993). "Nitrogen Isotope Techniques." Academic Press, San Diego.

Levin, M. A., Seidler, R. J., and Rogul, M. (1992). "Microbial Ecology: Principles, Methods and Applications." McGraw-Hill, New York.

Maier, R. M., Pepper, I. L., and Gerba, C. P., eds. (2000). "Environmental Microbiology." Academic Press, San Diego.

Marx, M. C., Wood, M., and Jarvis, S. C. (2001). A microplate fluorimetric assay for the study of enzyme diversity in soils. Soil Biol. Biochem. 33, 1633-1640.

McCarthy, A. J., and Williams, S. T. (1991). Methods for studying the ecology of actinomycetes. In "Methods in Microbiology" (R. Grigorova and J. R. Norris, eds.), Vol. 22, pp. 533-563. Academic Press, London.

Murphy, D. V., Recous, S., Stockdale, E. A., Fillery, I. R. P., Jensen, L. S., Hatch, D. J., and Goulding, K. W. T. (2003). Gross nitrogen fluxes in soil: theory, measurement and application of N-15 pool dilution techniques. Adv. Agron. 79, 69-118.

Nannipieri, P. (1994). Enzyme activity. In "The Encyclopaedia of Soil Science and Technology" (C. W. Finke, Jr., ed.). Van Nostrand Reinhold, New York.

Nannipieri, P., Kandeler, E., and Ruggiero, P. (2002). Enzyme activities and microbiological and biochemical processes in soil. In "Enzymes in the Environment—Activity, Ecology and Applications" (R. G. Burns and R. P. Dick, eds.), pp. 1-34. Dekker, New York.

Newell, S. Y., and Fallon, R. D. (1991). Toward a method for measuring instantaneous fungal growth rates in field samples. Ecology 72, 1547-1559.

Nylund, J. E., and Wallander, H. (1992). Ergosterol analysis as means of quantifying mycorrhizal biomass. Methods Microbiol. 24, 77-88.

Papale, D., and Valentini, R. (2003). A new assessment of European forests carbon exchange by eddy fluxes and artificial neural network spatialization. Global Change Biol. 9, 525-535.

Potthoff, M., Loftfield, N., Buegger, F., Wick, B., Jahn, B., Joergensen, R. G., and Flessa, H. (2003). The determination of 613C in soil microbial biomass using fumigation-extraction. Soil Biol. Biochem. 35, 947-954.

Preston-Mafham, J., Boddy, L., and Randerson, P. F. (2002). Analysis of microbial community functional diversity using sole-carbon-source utilisation profiles—a critique. FEMS Microbiol. Ecol. 42, 1-14.

Robertson, J. P. (1994). The impact of soil and crop management practices on soil spatial heterogeneity. In "Soil Biota" (C. E. Pankhurst, B. M. Doube, V. Gupta, and P. Grace, eds.), pp. 156-161. CSIRO, East Melbourne.

Robertson, G. P., Coleman, D. C., Bledsoe, C. S., and Sollins, P. (1999). "Standard Soil Methods for Long-Term Ecological Research." Oxford Univ. Press, New York.

Ruess, L., Haggblom, M. M., Langel, R., and Scheu, S. (2004). Nitrogen isotope ratios and fatty acid composition as indicators of animal diets in belowground systems. Oecologia 139, 336-346.

Schimel, D. S. (1993). "Theory and Application of Tracers." Academic Press, San Diego.

Schinner, F., Ohlinger, R., Kandeler, E., and Margesin, R., eds. (1996). "Methods in Soil Biology." Springer, Berlin.

Sinsabaugh, R. L., Carreiro, M. M., and Alvarez, S. (2002). Enzymes and microbial dynamics of litter decomposition. In "Enzymes in the Environment—Activity, Ecology, and Applications." (R. G. Burns and R. P. Dick, eds.), pp. 249-266. Dekker, New York.

Stemmer, M. (2004). Multiple-substrate enzyme assays: a useful approach for profiling enzyme activity in soils? Soil Biol. Biochem. 36, 519-527.

Stotzky, G., and Bollag J.-M., eds. (1996). "Soil Biochemistry." Vol. 9. Dekker, New York.

Tabatabai, M. A. (1994). Soil enzymes. In "Methods of Soil Analysis," Part 2, "Microbiological and Biochemical Properties" (R. W. Weaver, J. S. Angel, and P. S. Bottomley, eds.), pp. 775-833. Soil Sci. Soc. of America, Madison, WI.

Tabatabai, M., and Fung, M. (1992). Extraction of enzymes from soil. In "Soil Biochemistry" (G. Stotzky and J.-M. Bollag, eds.), Vol. 7, pp. 197-227. Dekker, New York.

Tabatabai, M. A., and Dick, W. A. (2002). Enzymes in soil—research and development in measuring activities. In "Enzymes in the Environment—Activity, Ecology and Applications" (R. G. Burns and R. P. Dick, eds.), pp. 567-596. Dekker, New York.

Tunlid, A., and White, D. C. (1992). Biochemical analysis of biomass, community structure, nutritional status, and metabolic activity of microbial communities in soil. In "Soil Biochemistry" (G. Stotzky and J.-M. Bollag, eds.), Vol. 7, pp. 229-262. Dekker, New York.

Vepsalainen, M., Kukkonen, S., Vestberg, M., Sirvió, H., and Niemi, R. M. (2001). Application of soil enzyme activity test kit in a field experiment. Soil Biol. Biochem. 33, 1665-1672.

Verma, B., Robarts, R. D., Headley, J. V., Peru, K. M., and Christofi, N. (2002). Extraction efficiencies and determination of ergosterol in a variety of environmental matrices. Commun. Soil Sci. Plant Anal. 33, 3261-3275.

Vestal, J. R., and White, D. C. (1989). Lipid analysis in microbial ecology. Bioscience 39, 535-541.

Weaver, R., Angle, S., and Bottomley, P. (1994). "Methods of Soil Analyses," Part 2, "Biochemical and Biological Properties of Soil." Am. Soc. Agronomy, Madison, WI.

Wilkinson, M. H. F., and Schut, F., eds. (1998). "Digital Image Analysis of Microbes—Imaging, Morphology, Fluorometry and Motility Techniques and Applications." Wiley, Chichester.

Wright, S. F. (2000). A fluorescent antibody assay for hyphae and glomalin from arbuscular mycorrhizal fungi. Plant Soil 226, 171-177.

Was this article helpful?

0 0
Organic Gardeners Composting

Organic Gardeners Composting

Have you always wanted to grow your own vegetables but didn't know what to do? Here are the best tips on how to become a true and envied organic gardner.

Get My Free Ebook


Post a comment