Construction of a rhizosphere pseudomonad with potential to degrade polychlorinated biphenyls and detection of bph gene expression in the rhizosphere

Abstract

The genetically engineered transposon TnPCB, contains genes (bph) encoding the biphenyl degradative pathway. TnPCB was stably inserted into the chromosome of two different rhizosphere pseudomonads. One genetically modified strain, Pseudomonas fluorescens F113pcb, was characterized in detail and found to be unaltered in important parameters such as growth rate and production of secondary metabolites. The expression of the heterologous bph genes in F113pcb was confirmed by the ability of the genetically modified microorganism to utilize biphenyl as a sole carbon source. The introduced trait remained stable in laboratory experiments, and no bph-negative isolates were found after extensive subculture in nonselective media. The bph trait was also stable in nonselective rhizosphere microcosms. Rhizosphere competence of the modified F113pcb was assessed in colonization experiments in nonsterile soil microcosms on sugar beet seedling roots. F113pcb was able to colonize as efficiently as a marked wild-type strain, and no decrease in competitiveness was observed. In situ expression of the bph genes in F113pcb was found when F113pcb bearing a bphlacZ reporter fusion was inoculated onto sugar beet seeds. This indicates that the bph gene products may also be present under in situ conditions. These experiments demonstrated that rhizosphere-adapted microbes can be genetically manipulated to metabolize novel compounds without affecting their ecological competence. Expression of the introduced genes can be detected in the rhizosphere, indicating considerable potential for the manipulation of the rhizosphere as a self-sustaining biofilm for the bioremediation of pollutants in soil. Rhizosphere bacteria such as fluorescent Pseudomonas spp. are ecologically adapted to colonize and compete in the rhizosphere environment. Expanding the metabolic functions of such pseudomonads to degrade pollutants may prove to be a useful strategy for bioremediation.

A bioluminescent derivative of Pseudomonas putida KT2440 for deliberate release into the environment

“…The so-called £uorescent Pseudomonas group includes strains whose biochemical, physiological and genetic properties have been well characterized. A number of genetic tools^broad host range vectors, transposons and minitransposons, reverse genetics etc.^have made it possible to design recombinant derivatives of this group of bacteria for use as microbial inoculants in agriculture for the biological control of soil-borne pests [1^3], for promoting plant growth [4,5], or for plant-assisted microbial elimination of pollutants [6,7].…”

Mini-transposons in microbial ecology and environmental biotechnology

“…The use of mini-transposons is, therefore, optimal in metabolic engineering projects that require a high predictability of the biocatalyst. Examples of this kind include the construction of surfactant-producing Pseudomonas strains inserted with a gene cassette from Rhodococcus for desulfurisation of dibenzothiophene [54] as well as the production of root-colonizing, polychlorobiphenyl (PCB)-degrader recombinant Pseudomonas for rhizo-remediation of soil polluted with these chloroaromatic compounds [55].…”

Fluorescence Resonance Energy Transfer (FRET) based molecular detection of a genetically modified PCB degrader in soil

“…One of the most promising bioremediation strategies is rhizoremediation which involves the use of plants and rhizosphere microorganisms (for recent review see [6]). Bacterial colonisation of plant roots is a complex process and a possible approach to improve rhizoremediation is to introduce degradation genes into good root colonisers, for example, TCE degradation [7] and PCB degradation [8]. The genetically modified microorganism (GMM), Pseudomonas fluorescens strain F113rifPCB, was constructed by introducing the bph LB400 operon (for biphenyl and PCB co-metabolism) into the chromosome of the root colonising strain F113.…”