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 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.
Full Text
The Full Text of this article is available as a PDF (241.8 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barriault D., Sylvestre M. Factors affecting PCB degradation by an implanted bacterial strain in soil microcosms. Can J Microbiol. 1993 Jun;39(6):594–602. doi: 10.1139/m93-086. [DOI] [PubMed] [Google Scholar]
- Bedard D. L., Unterman R., Bopp L. H., Brennan M. J., Haberl M. L., Johnson C. Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls. Appl Environ Microbiol. 1986 Apr;51(4):761–768. doi: 10.1128/aem.51.4.761-768.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dowling D. N., Pipke R., Dwyer D. F. A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs). FEMS Microbiol Lett. 1993 Oct 15;113(2):149–154. doi: 10.1111/j.1574-6968.1993.tb06506.x. [DOI] [PubMed] [Google Scholar]
- Eltis L. D., Hofmann B., Hecht H. J., Lünsdorf H., Timmis K. N. Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem. 1993 Feb 5;268(4):2727–2732. [PubMed] [Google Scholar]
- Erickson B. D., Mondello F. J. Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol. 1992 May;174(9):2903–2912. doi: 10.1128/jb.174.9.2903-2912.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fenton A. M., Stephens P. M., Crowley J., O'Callaghan M., O'Gara F. Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl Environ Microbiol. 1992 Dec;58(12):3873–3878. doi: 10.1128/aem.58.12.3873-3878.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goerlitz D. F., Law L. M. Determination of chlorinated insecticides in suspended sediment and bottom material. J Assoc Off Anal Chem. 1974 Jan;57(1):176–181. [PubMed] [Google Scholar]
- Hsu T. S., Bartha R. Accelerated mineralization of two organophosphate insecticides in the rhizosphere. Appl Environ Microbiol. 1979 Jan;37(1):36–41. doi: 10.1128/aem.37.1.36-41.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lajoie C. A., Zylstra G. J., DeFlaun M. F., Strom P. F. Development of field application vectors for bioremediation of soils contaminated with polychlorinated biphenyls. Appl Environ Microbiol. 1993 Jun;59(6):1735–1741. doi: 10.1128/aem.59.6.1735-1741.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lappin H. M., Greaves M. P., Slater J. H. Degradation of the herbicide mecoprop [2-(2-methyl-4-chlorophenoxy)propionic Acid] by a synergistic microbial community. Appl Environ Microbiol. 1985 Feb;49(2):429–433. doi: 10.1128/aem.49.2.429-433.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Unterman R., Bedard D. L., Brennan M. J., Bopp L. H., Mondello F. J., Brooks R. E., Mobley D. P., McDermott J. B., Schwartz C. C., Dietrich D. K. Biological approaches for polychlorinated biphenyl degradation. Basic Life Sci. 1988;45:253–269. doi: 10.1007/978-1-4899-0824-7_17. [DOI] [PubMed] [Google Scholar]
- Voisard C., Keel C., Haas D., Dèfago G. Cyanide production by Pseudomonas fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions. EMBO J. 1989 Feb;8(2):351–358. doi: 10.1002/j.1460-2075.1989.tb03384.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Walton B. T., Anderson T. A. Microbial degradation of trichloroethylene in the rhizosphere: potential application to biological remediation of waste sites. Appl Environ Microbiol. 1990 Apr;56(4):1012–1016. doi: 10.1128/aem.56.4.1012-1016.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]