plant surface microbiology.pdf

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Anton Hartmann et al.
root surface colonization by a particular bacterial strain could be investigated
in a background of other members of this species, identified by using rRNAtargeting
probes and FISH.
2.3 Application of Fluorescence Tagging and Reporter Constructs
The fate of particular bacterial inocula in the rhizosphere can also be monitored
using molecular-tagged bacteria. In addition to the use of the visually
detectable lux- and gus-markers (Lux: luciferase, Gus: b-glucuronidase), the
exploitation of the green fluorescent protein (GFP) from the jellyfish
Aequorea victoria has brought further progress into the field. GFP is a protein
that contains a fluorescent cyclic tripeptide sequence. It requires only molecular
oxygen for fluorescence, which means that GFP will fluoresce in virtually
any aerobic organism (Lorang et al. 2001). Therefore, GFP-labeled bacteria
can be observed by CLSM or by regular fluorescence microscopy. Figure 2C, D
shows a localization of GFP-labeled Serratia liquefaciens MG44 in the rhizoplane
of tomato. Furthermore, the application of DsRed from Discosoma sp.
provides a red fluorescing molecular marker (Christensen et al. 1999; Tolker-
Nielsen et al. 2000). In addition, a mutated form of GFP (ASV) with a short
half-life enables real-time in situ expression studies (Andersen et al. 1998;
Ramos et al. 2000).
The application of GFP-labeling in expression studies using promotor-gfp
fusions and GFP fusion proteins has revolutionized the in situ activity studies,
because of the relative ease of recording the fluorescence microscopically.
The bacteria carrying the gene constructs either on a plasmid or integrated
into the chromosome are applied to sense or report conditions in the microhabitat
they have been introduced. As in the case of simple tagging of organisms,
not only lux- and gus-reporter (Kragelund et al. 1997) were used, but
also constructs using the ice-nucleation gene (Loper and Henkels 1997), or
the ferrichrom iron receptor (FhuA; Stubner et al. 1994). These constructs
allowed the in situ sensing of N-, P- and C-starvation response (Kragelund et
al. 1997; Koch et al. 2001), expression of nitrogen fixation genes (Egener et al.
1999), presence of oxygen (Hojberg et al. 1999), availability of iron (Loper and
Henkels 1997) general activity and cell number (Unge et al. 1999), genotoxic
effects (Stubner et al. 1994) or the presence of quorum-sensing signal molecules
of the N-acylhomoserine lactone type (Steidle et al. 2001). Figure 2C
provides an example of in situ localization of GFP-labeled Serratia liquefaciens
MG44 on root hairs in the rhizosphere of tomato as a bright field picture
with 488-nm excitation wavelength, while Fig. 2D shows the same sample as
CLSM-picture with two excitation wavelengths (560 and 488 nm) making the
RFP-labeled Pseudomonas putida isoF also visible.
In some of these studies, bacterial cells with reporter constructs need to
be extracted from the habitat for analysis (Koch et al. 2001). Although these