As illustrated above, bacteria marked with GFP can dearly be visualized in situ, allowing identification of the environmental niche they occupy. One research area that has greatly benefited from the use of GFP has been the localization of plant-associated bacteria. An example of the use of GFP as a marker to localize specific bacteria directly on plant surfaces is shown in Fig. 7.1. In this study, the biocontrol strain P. chlororaphis MA 342, which controls fungal disease on cereal crops, was chromosomally tagged with two copies of the Gfp gene to enhance GFP fluorescence, and enabling visualization of the pattern of colonization of the cells on barley seeds by fluorescence stereomicroscopy and confocal microscopy (Fig. 7.1).49 We have also used bacteria marked with a transposon containing GFP to monitor the interactions between rice roots and endophytic bacteria (Stoltzfus and de Bruijn, unpublished data). Moreover, Gage et al used confocal microscopy to monitor early events in rhizobial infection and nodule formation on alfalfa using aSinorhizobium meliloti isolate carrying a plasmid constitutively expressing GFP34 and Bloemberg et. al used a combination of phase contrast and fluorescence microscopy to distinguish Pseudomonas aeruginosa cells containing plasmid-bome GFP from untagged Burkholderia cepacia bacteria attached to an abiotic surface.50 These authors also monitored GFP-tagged P. fluorescens bacteria associated with the roots of tomato seedlings by fluorescence microscopy.
There are several other published examples of the use of GFP as a marker for in situ localization of bacteria in other kinds of environments. For example, GFP-marked E. coli and Serratia marcescens cells have also been used to study flocculation in activated sludge by
Fig. 7.1. Confocal microscope projection of a stack of images of individual GFP-tagged Pseudomonas chlororaphis MA342G2 cells (white spots on this figure) distributed on the outer surface of a barley seed coat or glume (large cells). A model LSM501 laser scanning confocal microscope (Carl Zeiss, Jena, Germany) was used. The images are the result of pseudocolor merging of the output of three channels. Three-dimensional rendering of the stack of images was obtained by using the software 3D for LSM510, version 1.4 (Carl Zeiss).
confocal microscopy.18 In addition, Mycobacterium bovis cells were marked with GFP to follow their infection of mice cells using flow cytometry.30,51 The infection of human macrophages and epithelial cells by Mycobacterium avium marked with GFP has also been observed,52 and Valdivia et al used GFP to visualize infection of live mammalian cells by Salmonella typhimurium, Yersinia pseudotuberculosis and Mycobacterium marinum.5'3
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