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Application of Phenotypic Microarrays to Environmental Microbiology

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Application of Phenotypic Microarrays to Environmental Microbiology Available online at www.sciencedirect.com Application of phenotypic microarrays to environmental microbiology 1 1 1,2,3 Sharon Borglin , Dominique Joyner , Kristen M DeAngelis , 3 3,4 5,6 Jane Khudyakov , Patrik D’haeseleer , Marcin P Joachimiak and 1,3,7,8 Terry Hazen Environmental organisms are extremely diverse and only a extremes in pH, redox potential, substrate concentrations, small fraction has been successfully cultured in the laboratory. pressure, and temperature. Owing to the sometimes extre- Culture in micro wells provides a method for rapid screening of mely specific growth requirements of microbes, the use of a wide variety of growth conditions and commercially available multiwell plates for cultivation of distinct organisms has plates contain a large number of substrates, nutrient sources, become well-established for both bacteria and fungi and inhibitors, which can provide an assessment of the encompassing a wide array of applications in the environ- phenotype of an organism. This review describes applications mental, food safety, and medical fields. of phenotype arrays to anaerobic and thermophilic microorganisms, use of the plates in stress response studies, in Phenotypic microarray technology is growth of microor- development of culture media for newly discovered strains, and ganisms, either pure culture, community, or consortia, in for assessment of phenotype of environmental communities. multi-well plate each with a different test component in Also discussed are considerations and challenges in data each well, enabling a screen of the phenotypic character- interpretation and visualization, including data normalization, istics of the test culture. This technology allows for cultur- statistics, and curve fitting. ing in small volumes providing opportunity to perform Addresses many parallel assays in a compact space. Examples of well 1 Ecology Department, Earth Sciences Division, Lawrence Berkeley components could include but are not limited to C,N,P,S- National Laboratory, 1 Cyclotron Road, MS 70A-3317, Berkeley, CA sources, amino acids, pH or NaCl tolerance, or antibiotic 94720, USA 2 resistance. A phenotypic microarray is commercially avail- Microbiology Department, University of Massachusetts, Amherst, MA, USA able through Biolog, Inc. (Hayward, CA), which provides a 3 Microbial Communities Group, Joint Bioenergy Institute, Emeryville, suite of twenty 96-well plates, each containing a different CA, USA substrate, stressor, or nutrient [1,2]. This system works by 4 Biosciences and Biotechnology Division, Lawrence Livermore National culturing cells either aerobically or anaerobically [3,4], and Laboratory, Livermore, CA, USA 5 assaying growth colometrically on a microplate reader or Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA the Omnilog unit (Biolog, Hayward, CA). By comparing 6 Virtual Institute of Microbial Stress and Survival, Lawrence Berkeley growth in these plates, phenotypic changes can be detected National Laboratory, Berkeley, CA, USA as strains and environmental conditions are varied. The 7 Department of Civil and Environmental Engineering, University of plates can also be user prepared, giving greater control and Tennessee, 676 Dabney Hall, Knoxville, TN, 37996-1605, USA 8 knowledge of media components. Oak Ridge National Laboratory, Biosciences Division, Oak Ridge, TN, USA Key advances in the last few years have extended the Corresponding author: Hazen, Terry ([email protected]) application of growth phenotype arrays to organisms with special growth requirements. Several publications in Current Opinion in Biotechnology 2012, 23:41–48 recent years have described novel applications of pheno- typic microarrays that pave the way towards understanding This review comes from a themed issue on Analytical biotechnology environmental microbiology. Some of the most promising Edited by Wei E. Huang and Jizhong Zhou applications lie in defining changes in phenotypes as a result of genetic manipulation and screening for possible Available online 2nd January 2012 growth conditions for uncultured organisms. Several pub- 0958-1669/$ – see front matter lications have discussed the use of phenotypic microarrays Published by Elsevier Ltd. to assess phenotypes of knockout mutants [5 ,6 ,7,8,9 ,10] or investigation of chemotactic response [11 ]. Other appli- DOI 10.1016/j.copbio.2011.12.006 cations use microplates to investigate phenotypic changes owing to different environmental conditions such as temperature, antibiotic resistance, or other stressors Introduction [12 ,13 ,14,15 ]. Growth microarrays have also been used Growth conditions for microbes are as diverse as the to differentiate function between strains of the same environments they inhabit. Microbes have been found species [16]. These applications emphasize the need to in all investigated environments so far, which include confirm genetic predictions with phenotypic profiling. www.sciencedirect.com Current Opinion in Biotechnology 2012, 23:41–48 42 Analytical biotechnology In this review, several novel applications of the pheno- chamber and incubated anaerobically. The gas and plate typic microarrays are described, along with particular filled bags are seated in place to be read on the PM with a method modifications needed. Most current applications simple engineering of the trays with clamps. require the development of proper inoculation, growth conditions, and assay methods, which are necessary for When using byproducts of growth, such as precipitates reproducible and meaningful results. Also included is a generated by DvH or color change of the tetrazolium discussion of current data visualization and data proces- dyes, it must be noted that the assay is a measure of sing techniques, both of which present challenges sulfate reduction or other metabolic activity, which is encountered when interpreting PM data. possibly a byproduct of growth [22 ,23]. In addition there can be significant color change detected by reactions that Anaerobic growth can and do occur abiotically by some of the chemical The PM system was originally designed to profile meta- agents added to the PM wells [3]. This must be taken into bolic activity of aerobic organisms. To assay phenotypes account when discussing a kinetic plot as they are not all during aerobic metabolism a tetrazolium salt is used that comparable to traditional growth curves. Abiotic reactions is reduced by dehydrogenases and reductases produced can be assayed by filling wells with sterile or heat-killed by cells to yield a formazan dye, indicating that the medium. inoculated organism are actively metabolizing a substrate in the well. No color change implies that the cells are not Alternatively, other assays of growth or metabolism can be active and the substrate of interest, for example, a specific used. With the iron reducing anaerobe Geobacter metallir- carbon source, is not metabolized. For anaerobes, educens a metabolic dye could not be used as it was reduction of the medium is often required and the lower reduced by the medium and the turbidity of the final reduction potential of the media results in abiotic trans- biomass yield was too low to be detected by the Omnilog formation of the tetrazolium salts. However, studies have optics [24]. For this Fe reducer, the metabolism of the demonstrated that formazan dyes can be utilized to organism on substrates of interest was measured by a measure activity in growth assays under anaerobic con- colorimetric method utilizing ferrozine to quantify ditions [17,18] if the organism of interest can grow in non- reduced iron in the wells of the PM plate. With this reduced medium or if the added reductant does not lower method, the PM plates were inoculated and incubated, the redox potential below that required to transform the but not recorded by the data logging system. Each well indicator dye. However in these cases it is possible the was assayed destructively after incubating for a prescribed dyes will also not be reduced by the activity of the amount of time. In this case the Omnilog can only be used metabolizing organism and it may become necessary to as an endpoint detector. These assays could potentially add metabolic boosters such as yeast extract and be made high throughput by coupling the Omnilog with increased inoculum concentration to measure activity robotics systems (see Figure 1). of the organism [17,19]. Care should be taken when choosing the type of dye used, as some measure inter- Thermophilic growth cellular and others extra cellular activity, formazan pro- The Omnilog instrument has an operating incubating duced can be water soluble or insoluble, and some dyes temperature with a maximum of 45 8C. In the case of can cause cell death on transformation [20]. hyperthermophiles such as Sulfolobus sulfotaricus, high temperature phenotypic profiles were generated using Since the Omnilog detector measures opacity, altern- the PM array in a specialized incubator. Plates were atives can be found to dyes, usually by finding an indicator incubated at 80 8C without deformation of plastic or of activity that increases opacity. For the anaerobic sulfate destabilization of the dye. It is essential that the panels reducer Desulfovibrio vulgaris (DvH) the medium was be placed inside a humidified chamber to prevent evap- reduced with titanium citrate resulting in an electric oration. Activity was then logged as an endpoint or over potential of approximately À400 mV, which reduced all time on the Omnilog or other plate reader by
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