Final Report: Detection, Quantification, and Biogeography of Microbes in Ecosystems

EPA Grant Number: R823845
Title: Detection, Quantification, and Biogeography of Microbes in Ecosystems
Investigators: Andrews, John H.
Institution: University of Wisconsin Madison
EPA Project Officer: Hahn, Intaek
Project Period: September 28, 1995 through September 27, 1998
Project Amount: $401,130
RFA: Exploratory Research - Environmental Biology (1995) RFA Text |  Recipients Lists
Research Category: Biology/Life Sciences , Health , Ecosystems


To further develop and implement fluorescent nucleotide probes based on the 18S rRNA gene in FISH (fluorescence in situ hybridization) reactions for the yeast-like epiphytic fungus Aureobasididum pullulans; to install a bioluminescent marker system in A. pullulans based on the green fluorescent protein (GFP) system from the jellyfish Aequorea victoria; and to use the marker to test hypotheses related to colonization and emigration.

Summary/Accomplishments (Outputs/Outcomes):

To provide a means of specifically detecting Aureobasidium pullulans, Ap, we designed probes based on the 18S rRNA gene even though it contains fewer variable regions for distinguishing among closely related taxa than does the 28S rRNA gene. The former has been the molecule of choice in developing molecular phylogenies, and, accordingly, most of the sequence information published to date relates to this molecule. The taxonomy of Ap is uncertain, so sequence conservation among purported "strains" could not be presumed. Accordingly, the nucleotide sequence of 11 strains within a 578-bp variable region of the gene was determined. We had previously sequenced the entire gene to lay the groundwork for this approach. Extreme sequence conservation was observed. The consensus Ap sequence was compared with other fungal sequences to identify potential probes. A 21-mer probe which hybridized to the 12 Ap strains but not to 98 other fungi, including 82 isolates from the phylloplane, was identified. A 17-mer highly specific for Cladosporium herbarum also was identified.

The 21-mer oligonucleotide probe designated Ap665 was labelled with five molecules of fluorescein isothiocyanate, and applied by fluorescence in situ hybridization (FISH) to populations of the fungus on slides and apple leaves from growth chamber seedlings and orchard trees (Figs. 3,4) summarized as follows. In specificity tests that included Ap665 and a similarly labelled universal probe and the respective complementary probes as controls, the hybridization signal was strong for Ap665 reactions with 12 Ap strains but at or below background level for the 98 other fungi including 82 phylloplane isolates noted above. Scanning confocal laser microscopy was used to confirm that the fluorescence originated from the cytoplasmic matrix and to overcome limitations imposed on conventional microscopy by leaf topography. Images were recorded with a cooled charge-coupled device video camera and digitized for storage and manipulation. Image analysis was used to verify semiquantitative fluorescence ratings and to demonstrate how the distribution of the fluorescence signal in specific interactions (e.g., Ap665 with Ap cells) could be separated at a given probability level from nonspecific fluorescence (e.g., in interactions of Ap665 with Cryptococcus laurentii cells) of an overlapping population. Image analysis methods were used also to quantify epiphytic Ap populations based on cell number or percent coverage of the leaf surface. We have begun to implement this technology by enumerating populations along leaf transects (Fig. 4) of apple leaves collected at monthly intervals from orchard trees.

We have also developed a live/dead viability assay based on fluorochromes (Fig. 3). This has involved refining various techniques of vital staining; the most promising are based on the principle of a live stain using a fluorescent esterase substrate such as BCECF or Calcein blue AM (Molecular Probes). Both of these have better retention in cells than fluorescein diacetate, allowing some time to pass between staining and examination. Dead cells are stained with a dye that can penetrate compromised cell membranes such as propidium iodide or the DEAD Red component of the Live/Dead Reduced Biohazard Viability/Cytotoxicity Kit? also from Molecular Probes. The significance of our work here is that reliable microbial live/dead stains have traditionally been difficult to develop and those in existence have been restricted largely to common yeast, Saccharomyces cerevisiae, as a model. We have had to devise a viability stain for our own fungus and, moreover, demonstrate that it can be used in situ on leaf surfaces (rather than just on microscope slides as is typically the case).

Finally, we developed a bioluminescent tag for Ap based on the green fluorescent protein (GFP) from a jellyfish, Aequorea victoria (Figs. 1,2). This will enable transformed, GFP- expressing cells of Ap to be released to leaves and monitored over time for survival and growth, without confounding these cells with the wild-type (nonfluorescent) background population naturally present on the phylloplane. These studies have been approved by the campus biosafety committee, the State of Wisconsin, and Federal authorities (USDA APHIS) and are in progress (summer and fall 1998). In 1994, Chalfie et al showed that a jellyfish cDNA encoding GFP could be expressed in E. coli and Caenorhabditis elegans. Expression of wild-type or enhanced, mutated forms of GFP has since been demonstrated in numerous organisms. Subsequently, GFP has been developed as a reporter for gene expression, a marker of subcellular protein localization, a tracer of cell lineage and a label to follow development of pathogens within their plant hosts. As a marker system, especially for living cells, GFP has several advantages over existing reporters such as -glucuronidase (GUS), chloroamphenicol acetyltransferase (CAT), - galactosidase (LacZ) or luciferase (LUC). These include: (i) its stability (little or no photobleaching); (ii) ability to form fluorescent, functional products with other proteins; and (iii) simplicity and versatility for in vivo use (requiring only blue or UV light and oxygen to fluoresce.

Our strategy for expressing GFP involved fusing a highly expressed, constitutive promoter (the 800-bp translation elongation factor from Ap) with an improved GFP cDNA and then stably introducing the expression vector into Ap by co-transformation with pDH33, a plasmid conferring hygromycin resistance. We obtained three transformants (and subsequently several others) and confirmed expression visually, by Southern blots, and by absorption spectra (Figs.1,2). Our report was the second ever for GFP expression in a filamentous fungus and the work was featured twice, by invitation, on the cover of BioTechniques (Fig. 2). As is evident from the figures, GFP-Ap is readily detectable on the phylloplane of apple seedlings from growth chambers or orchard trees and amenable to quantification by image analysis. We are now awaiting federal authorization to introduce GFP-tagged cells to the orchard this summer. Preliminary experiments to test hypotheses about microbial community assembly on leaves and on the role of emigration in determining epiphytic population size are in progress.

Journal Articles on this Report : 4 Displayed | Download in RIS Format

Other project views: All 8 publications 4 publications in selected types All 4 journal articles
Type Citation Project Document Sources
Journal Article Li S, Spear RN, Andrews JH. Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscope slides and leaf surfaces. Applied Environmental Microbiology 1997;63(8):3261-3267. R823845 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Other: AEM PDF
  • Journal Article Spear RN, Li S, Nordheim EV, Andrews JH. Quantitative imaging and statistical analysis of fluorescence in situ hybridization (FISH) of Aureobasidium pullulans. Journal of Microbiological Methods 1999;35(2):101-110. R823845 (Final)
  • Abstract from PubMed
  • Other: Science Direct PDF
  • Journal Article Spear RN, Cullen D, Andrews JH. Fluorescent labels, confocal microscopy, and quantitative image analysis in the study of fungal biology. Methods in Enzymology 1999;307(34):607-623. R823845 (Final)
  • Abstract from PubMed
  • Other: FPL PDF
  • Journal Article VandenWymelenberg AJ, Cullen D, Spear RN, Schoenike B, Andrews JH. Expression of green fluorescent protein in Aureobasidium pullulans and quantification of the fungus on leaf surfaces. Biotechniques 1997;23(4):686-690. R823845 (Final)
  • Abstract from PubMed
  • Other: FPL PDF
  • Supplemental Keywords:

    assessment; terrestrial; phylloplane; phyllosphere; microbiology; epifluorescence; probe; fungi; yeast; GFP; 18S rRNA fluorescence; microscopy; bioluminescent; marker, RFA, Scientific Discipline, Ecosystem Protection/Environmental Exposure & Risk, exploratory research environmental biology, Environmental Chemistry, Ecosystem/Assessment/Indicators, Chemical Mixtures - Environmental Exposure & Risk, Ecosystem Protection, Chemistry, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Ecological Indicators, bioindicator, bioluminescent marker, biogeochemical indicators, quantification of microbes, aureobasidium pullulans, jellyfish

    Progress and Final Reports:

    Original Abstract
  • 1996
  • 1997