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

• Published in: Applied and Environmental Microbiology 1995-05, Vol.61 (5), p.1946-1952
• Main Authors: Brazil, G M, Kenefick, L, Callanan, M, Haro, A, de Lorenzo, V, Dowling, D N, O'Gara, F
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; beta vulgaris; bifenilos policlorados; biodegradacion; biodegradation; Biodegradation of pollutants; Biodegradation, Environmental; Biological and medical sciences; Biology; Biotechnology; Cloning, Molecular; control de la contaminacion; DNA Transposable Elements - genetics; Environment and pollution; expresion genica; expression des genes; Fundamental and applied biological sciences. Psychology; gene; gene expression; Gene Expression Regulation, Bacterial; gene transfer; Genes; Genetic Engineering; genetic transformation; Genetics; Industrial applications and implications. Economical aspects; inoculacion; inoculation; inoculation methods; lutte antipollution; Multienzyme Complexes - biosynthesis; Multienzyme Complexes - genetics; Operon; Oxidoreductases - biosynthesis; Oxidoreductases - genetics; Oxygenases - biosynthesis; Oxygenases - genetics; pollution control; polychlorinated biphenyls; Polychlorinated Biphenyls - metabolism; polychlorure de biphenyle; Pseudomonas fluorescens; Pseudomonas fluorescens - genetics; Pseudomonas fluorescens - growth & development; Pseudomonas fluorescens - metabolism; Recombinant Fusion Proteins - biosynthesis; rhizosphere; rizosfera; seed; semence; semillas; Soil Microbiology; Soil Pollutants - metabolism; transferencia de genes; transfert de gene; transformacion genetica; transformation genetique; Vegetables - microbiology
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.61.5.1946-1952.1995

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 bph'lacZ 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.