Population dynamics and gene transfer in genetically modified bacteria in a model microcosm.

The horizontal transfer and effects on host fitness of a neutral gene cassette inserted into three different genomic loci of a plant-colonizing pseudomonad was assessed in a model ecosystem. The KX reporter cassette (kanamycin resistance, aph, and catechol 2, 3, dioxygenase, xylE) was introduced on the disarmed transposon mini-Tn5 into: (I) the chromosome of a spontaneous rifampicin resistant mutant Pseudomonas fluorescens SBW25R; (II) the chromosome of SBW25R in the presence of a naturally occurring lysogenic-phage (phage Phi101); and (III) a naturally occurring plasmid pQBR11 (330 kbp, tra+, Hgr) introduced into SBW25R. These bacteria were applied to Stellaria media (chickweed) plants as seed dressings [c. 5 x 104 colony-forming units (cfu)/seed] and the seedlings planted in 16 microcosm chambers containing model plant and animal communities. Gene transfer to pseudomonads in the phyllosphere and rhizosphere was found only in the plasmid treatment (III). Bacteria in the phage treatment (II) initially declined in density and free phage was detected, but populations partly recovered as the plants matured. Surprisingly, bacteria in the chromosome insertion treatment (I) consistently achieved higher population densities than the unmanipulated control and other treatments. Plasmids were acquired from indigenous bacterial populations in the control and chromosome insertion treatments. Plasmid acquisition, plasmid transfer from inocula and selection for plasmid carrying inocula coincided with plant maturation.

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.

Strain characterization and 16S-23S probe development for differentiating geographically dispersed isolates of the phytopathogen Ralstonia solanacearum.

The causative agent of potato brown rot and bacterial wilt, Ralstonia solanacearum, results in serious world-wide economic losses, particularly in the tropics. In the last decade, however, the incidence of bacterial wilt in potatoes grown in Northern Europe has increased, presenting an interesting epidemiological puzzle. Its occurrence may be as a result of changes in agricultural practice or the emergence of a novel bacterial variety, better adapted to cooler conditions. To understand the distribution and genetic diversity of this phytopathogen, we have analysed a collection of 82 isolates from Europe and tropical regions. Both phenotypic [SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) profiling, FAME (fatty acid methyl esters) analysis, growth profiles and EPS (exopolysaccharide) production] and genotypic [16S rRNA RFLP (restriction fragment length polymorphism), ARDRA (amplified ribosomal DNA restriction analysis) and sequence analysis of 16S-23S rRNA ITS and flanking regions] methods were compared. Principal component analysis of FAME profiles clustered isolates into three groups and ARDRA of a 0.85 kb amplified fragment from the 16S-23S ITS region differentiated isolates into four groups. Using sequence analysis, specific primers were designed within the variable region 147-170 of the 23S rRNA. These primers, RsolT2 and RsolT3, respectively, differentiated isolates into two distinct clusters as described previously by Wullings and colleagues (Wullings et al., 1998). The European strains (Biovar 2, race 3) analysed in this study specifically hybridized with RsolT3, and showed considerable genetic homogeneity when compared with strains of other races from 'the rest of the world'. These data indicate the possible selection and proliferation of a 'European'-adapted variant.

Chromosomal insertion of phenazine-1-carboxylic acid biosynthetic pathway enhances efficacy of damping-off disease control by Pseudomonas fluorescens.

A disarmed Tn5 vector (pUT::Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P fluorescens::phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.

Immuno-capture differential display method (IDDM) for the detection of environmentally induced promoters in rhizobacteria.

A rapid immunological method for trapping and selection of functionally regulated prokaryotic promoters is described. The method is based on application of a novel mini-Tn5 derived promoter probe (pUTTKZY-promoterless lacZY as a reporter and kanamycin resistance) to mutagenise a plant growth promoting fluorescent pseudomonad, Pseudomonas fluorescens 54/96. The transposon allows selection of operon fusion mutants (lacZY(+)) directly on media containing lactose as a sole carbon source as well as selection for kanamycin and lacZ (beta-galactosidase) expression on X-gal indicator media. We have extended the technique to target the surface expression of the induced lactose permease gene (lacY) from mutagenised libraries and the immuno-capture of bacteria with magnetic beads and anti-LacY monospecifc antisera. The benefits of the lacZY reporter are that a library can be rapidly generated and screened in vitro to isolate non-expressed mutants for further in situ screening. Here we demonstrate the development and utility of the technique and its potential as a differential display method for the isolation of promoters that direct regulated gene expression in the phytosphere, or under other imposed conditions.

Identification of conserved traits in fluorescent pseudomonads with antifungal activity.

A collection of 29 fluorescent pseudomonads, some with known biological control activity against a range of phytopathogenic fungi, were characterized phenotypically and genotypically by comparing carbon source utilization patterns, suppression of Pythium ultimum both in planta and in vitro and the potential to produce known secondary metabolites. Fatty acid profiling and restriction fragment length polymorphism (RFLP) analysis of the ribosomal DNA operon (ribotyping) were used to determine the diversity of isolates. A small group of genetically related Pseudomonas spp. with similar properties was identified; each isolate produced a diffusible bioactive product in vitro and was active against Pythium ultimum in planta. However, other isolates that were able to suppress damping off disease but did not inhibit hyphal extension in vitro clustered outside this group. Phenotypic analyses revealed that the accumulation of C17:0 cyclopropane fatty acid (17CFA) and the production of hydrogen cyanide correlated significantly with biological control activity and with the antagonism of fungal development. The potential of 17CFA as a marker for the selection of fluorescent pseudomonads with biocontrol agent (BCA) potential was demonstrated by the isolation of a novel active strain. This was selected after the screening of 13 clonal groups of fluorescent pseudomonads identified from 500 isolates from the phytosphere of sugar beet. Levels of 17CFA synthesis possibly reflect the efficacy of the rpoS allele in particular strains.