4. Plant–microbe interactionsGage e

4. Plant–microbe interactions
Gage et al. (1996) labelled Rhizobium meliloti MB5OI with the gfp marker via the broad-host-range plasmid, pTB93F. The heterologous gfp was expressed constitutively in this microorganism. Visualization of the R. meliloti cells during infection of the root and subsequent nodulation was observed. The gfp system has also been used to visualize bacterial distributions on root surfaces (Fig. 1d). Bloemberg et al. (1997) constructed a gfp plasmid (pGBS) which was maintained in Pseudornonas fluorescens WC5365 cells for 7 days without antibiotic selective pressure. The gfp was constitutively expressed in the cells without nutrients being supplied. Under laboratory conditions, the gfp-labelled WCS36S cells were observed to form microcolonies at the borders of adjacent epithelial cells on the root surface of tomato seedlings. Pseudomonas fluorescens A506 has been marked chromosomally with a red-shift gfp variant (Tombolini et al., 1997). Because of the improved fluorescent intensity of the red-shift gfp and a strong constitutive promoter (PpsbA from Amaranthus hybridus), individual cells were visualized on the root surface of Lotus japonicus plants using epifluorescent confocal laser scanning microscopy.








5. Biofilms
The gfp system is ideal for studying multiplespecies bacterial communities in biofilms and adhesion of bacteria to flocs in activated sludge. Mo¨ller et al. (1998) demonstrated that gfp-labelled Pseudomonas putida RI tended to colonize the outermost layer of a mixed-species biofilm while an Acinetobacter sp. strain C6 primarily attached to the substratum of the biofilm. Skillman et al. (1998) visualized bacterial distribution in dual-species biofilms using a gfp-labelled Enterobacter. agglomerans strain and an unlabelled Kiebsiella pneumoniae strain. Sixteen-hour biofilms were stained using propidium iodide (P1) (did not mask the GFP), which enabled the reseachers to visualize the spatial distribution of the gfp-labelled cells in green or yellow, and the P1 stained cells as red using a violet-blue excitation filter (395–446 nm) on a microscope.

The two bacteria were often closely associated in microcolonies, and the reseachers concluded that surface-associated macromolecules form the basis of these interactions. They suggested that this convenient technique enabled them to observe these unique interactions between the bacteria and to determine the true properties of the resultant biofilm.
A gfp-labelled Pseudomonas putida has been used to study bacterial survival in activated sludge (Eberl et al., 1997). Free swimming cells were observed between sludge flocs immediately after their introduction and after 2 mm, protozoan grazing on the labelled cells was observed. After 3 days incubation, few fluorescent cells were detectable, but most of these cells were incorporated into the sludge flocs suggesting a protective role of the floc from predatory protozoa. The association between the GFP9 marked cells and flocs was visualized using epifluorescent microscopy. Using gfp-labelled E. coli and Serratia marcescens strains, Olofsson et al. (1998) determined that cell surface hydrophobicity degradwas an important factor in bacterial adhesion to flocs. Epifluorescent microscopy enabled the visualization and enumeration of free GFP marked cells which could be distinguished in the activated sludge. Confocal laser scanning microscopy (CLSM) of the gfp-
labelled bacteria enabled a 3-dimensional profile to be produced which showed that the two bacteria had very different attachment patterns in the flocs, and that cells can penetrate the flocs through channels and pores. The authors suggested the combination of epifluorescent and confocal laser scanning microscopy to visualize GFP-marked cells offers great potential for further investigations. Their approach resulted in a better understanding of bacterial adhesion mechanisms and may assist in improving the effectiveness of wastewater treatment.





6. Biodegradation
In many biodegradation studies in the laboratory, bacterial cells which are metabolically capable of degrading or mineralizing a pollutant are added to contaminated environmental samples to determine biodegradation
of target compound(s) in the environment. Cell counts, survival, metabolic activity, spatial location and degradative capability are all important factors that provide valuable information to advance our understanding of the processes occur ring. The use of the GFP as a marker has allowed researchers to detect the added cells, and determine more precise data about cell counts, survival and spatial location than has previously been possible. In our laboratory, the GFP marker was employed to study the survival of a p-nitrophenol
(PNP)-degrading Moraxella strain in soil. The chromosomally gfp-labelled Moraxella sp. strain G21 had been constructed using a mini-Tn5-gfp suicidal plasmid (see Fig. 2) (Tresse et al., 1998). The labeled bacteria degraded up to 1440 pM PNP, both in broth and soil, to the same extent as the parent strain. Survival studies indicated that individual green fluorescent colonies of G21 were detected up to 2 weeks after inoculation in soil samples. It is noteworthy that although only 0.1% of the original inoculum was detected by plating, 58% of the radiolabelled PNP was mineralized. This suggests that not all cells were culturable, or that there may have been an indigenous population of PNP-degrading microorganisms present in the soil. Errampalli et al. (1998) detected GFP-marked Pseudomonas sp. UGI4Gr up to 13 months after inoculation into creosote contaminated soil. By inserting the mini Tn5-gfp into the chromosome of a phenanthrenedegrading bacterium, a green fluorescent mutant, Pseudomonas sp. UGI4Gr, was obtained. The gfp was stable in UGi4Gr and did not affect mineralization of C-labelled phenanthrene which verified the gfp insertion had not interfered with phenan threne-degradation genes. Colony forming units were used to enumerate GFP-marked cells from soil samples and UG14Gr colonies on solid agar plates were detected using a hand-held UV lamp. Detection of GFP-marked cells for more than a year after inoculation into soil microcosms demonstrated the GFP marker can be useful for long-term bacterial survival in soil biodegradation or studies.