plant surface microbiology.pdf

23 Visualisation of Rhizosphere Interactions 439
methods that can be used to detect GFP-marked bacteria include the use of
charge couple device (CCD) microscopy and cell sorting by fluorescent-activated
cell sorters (FACS), which allows the sampling and identification of subpopulations
of bacteria in a non-destructive way at the single cell level. Autofluorescently
labelled colonies on agar plates can be detected under a
hand-held UV-lamp or a low-resolution binocular microscope equipped with
a UV lamp.
Since gfp is eukaryotic in origin, optimised constructs for the expression of
gfp in bacteria have been constructed and successfully applied. This was
achieved by expression of gfp under the control of strong constitutive promoters
or using red-shifted and UV-optimised mutant derivates. These GFP
variants provide an increased fluorescent signal intensity in bacteria, faster
rates of oxidative chromophore formation, resistance to photobleaching and
excitation maximums better suited to conventional detection instruments.
GFPuv emits bright green light (maximum at 509 nm) when exposed to UV or
blue light (395 or 470 nm). Mutant proteins GFPmut2 and GFPmut3 have
emission maximums of 507 and 511 nm when excited by blue light (481 and
501 nm, respectively).
Stable plasmid vectors (multicopy) and transposon vectors (single copy)
for marking with fluorescent proteins are available for use in Gram-negative
as well as Gram-positive bacteria. They can be used for tagging bacteria with
a biomarker, construction of fusion proteins, assaying gene activity, or promoter
probing. Plasmids pGB5, carrying gfp driven by a tac promoter, was
shown to be 100 % stably maintained in Pseudomonas in the tomato rhizosphere
and resulted in constitutive expression in Pseudomonas without addition
of an inducer. Dandie et al. (2001) constructed transposon-based tagging
vectors using a gfp marker gene under control of either constitutive or
inducible promoters.
Plasmids pFPV1 and pFPV2 direct high levels of gfp expression in E. coli,
Salmonella typhimurium, and Yersinia pseudotuberculosis and in different
mycobacterial species. The high levels of gfp expression were achieved by
expression under control of the lacZpo and hsp60 heat-shock promoters,
respectively. They have been used to visualise the infection process of mammalian
cells by the three species. Transposon plasmid Tn5GFP1 was successfully
used to follow Pseudomonas putida cells during water transport
through a sand matrix. To study the colonisation pattern of P. chlororaphis
MA342 on barley seeds, the strain was tagged using a plasmid pUTgfp2X
harbouring gfp.
For many applications, such as the analysis of chromosomal genes under
physiological (monocopy) conditions using transcriptional fusions, stable
integration of the reporter, or reduction of the risk of transfer of the genetic
marker to other microorganisms, it is necessary to integrate the gfp transcriptional
fusion into the chromosome of target bacteria by site-specific
recombination or by random insertion, e.g. by means of transposons. A gfp