Abstract
Mineralization of organic molecules by microbes is essential for the carbon cycle to operate. The massive mobilization of compounds stored in natural resources, or the introduction of xenobiotics into the biosphere, leads to unidirectional fluxes, which result in the persistance of a number of chemicals in the biosphere, and thus constitute a source of pollution. Molecular biology offers the tools to optimize the biodegradative capacities of microorganisms, accelerate the evolution of “new” activities, and construct totally “new” pathways through the assemblage of catabolic segments from different microbes. Although the number of genetically engineered microbes (GEMs) for potential use in biodegradation is not large, these recombinant microbes function in microcosms according to their design. The survival and fate of recombinant microbes in different ecological niches under laboratory conditions is similar to what has been observed for the unmodified parental strains. rDNA, both on plasmids and on the host chromosome, is usually stably inherited by GEMs. The potential lateral transfer of rDNA from the GEMs to other microbes is significantly diminished, though not totally inhibited, when rDNA is incorporated on the host chromosome. The behavior and fate of GEMs can be predicted more accurately through the coupling of regulatory circuits that control the expression of catabolic pathways to killing genes, so that the GEMs survive in polluted environments, but die when the target chemical is eliminated.
References
- 1.Keith LH, Telliard WA. Prioritary pollutants I—a perspective view. Environm. Sci. Technol. 1979;13:416–423. doi: 10.1021/es60152a601. [DOI] [Google Scholar]
- 2.Ramos JL, Timmis KN. Experimental evolution of catabolic pathways of bacteria. Microbiol. Sci. 1987;4:228–237. [PubMed] [Google Scholar]
- 3.Pemperton JM, Fisher PR. 2,4-D plasmids and persistence. Nature. 1977;268:732–733. doi: 10.1038/268732a0. [DOI] [PubMed] [Google Scholar]
- 4.Slater JH, Bull AT. Environmental microbiology: biodegradation. Phil. Trans. R. Soc. Lond. B. 1982;297:575–597. doi: 10.1098/rstb.1982.0063. [DOI] [Google Scholar]
- 5.Subba-Rao RV, Alexander M. Bacterial and fungal cometabolism of l,l,l-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) and its breakdown products. Appl. and Environm. Microbiol. 1985;49:509–516. doi: 10.1128/aem.49.3.509-516.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.De Lorenzo V, Timmis KN. Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5 and Tn10-derived mini-transposons. Methods Enzymol. 1994;235:386–405. doi: 10.1016/0076-6879(94)35157-0. [DOI] [PubMed] [Google Scholar]
- 7.Abril MA, Michán C, Timmis KN, Ramos JL. Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. J. Bacteriol. 1989;171:6782–6790. doi: 10.1128/jb.171.12.6782-6790.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Duque E, Haidour A, Godoy F, Ramos JL. Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene. J. Bacteriol. 1993;175:2278–2283. doi: 10.1128/jb.175.8.2278-2283.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Dowling DN, Pipke R, Dwyer DF. A DNA module encoding bph genes for the degradation of polychlorinated biphenyls (PCBs) FEMS Microbiol. Lett. 1993;113:149–154. doi: 10.1111/j.1574-6968.1993.tb06506.x. [DOI] [PubMed] [Google Scholar]
- 10.Lehrbach PR, Zeyer J, Reineke W, Knackmuss H-J, Timmis KN. Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13. J. Bacteriol. 1984;158:1025–1032. doi: 10.1128/jb.158.3.1025-1032.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Ramos JL, Stolz A, Reineke W, Timmis KN. Altered effector specificities in regulators of gene expression: TOL plasmid xylS mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. Proc. Natl. Acad. Sci. USA. 1986;83:8467–8471. doi: 10.1073/pnas.83.22.8467. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Ramos JL, Wasserfallen A, Rose K, Timmis KN. Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkyibenzoates. Science. 1987;235:593–596. doi: 10.1126/science.3468623. [DOI] [PubMed] [Google Scholar]
- 13.Reineke W, Knackmuss H-J. Construction of haloaromatic-utilizing bacteria. Nature. 1979;277:385–386. doi: 10.1038/277385a0. [DOI] [PubMed] [Google Scholar]
- 14.Rojo F, Pieper D, Engesser KH, Knackmuss H-J, Timmis KN. Assemblage of ortho cleavage routes for degradation of chloro-and methylaromatics. Science. 1987;238:1395–1398. doi: 10.1126/science.3479842. [DOI] [PubMed] [Google Scholar]
- 15.Sokacht J. The Biology of Pseudomonas. 1986. The Bacteria. [Google Scholar]
- 16.Galli E, Silver S, Withold B. Pseudomonas. Molecular Biology and Biotechnology. 1992. [Google Scholar]
- 17.Mermod N, Lehrbach PR, Don RH, Timmis KN. The Bacteria. 1986. Gene cloning and manipulation in Pseudomonas; pp. 325–355. [Google Scholar]
- 18.Worsey MJ, Williams PA. Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid. J. Bacteriol. 1974;124:7–13. doi: 10.1128/jb.124.1.7-13.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Dorn E, Hellwig M, Reineke W, Knackmuss H-J. Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad. Arch. Microbiol. 1974;99:61–70. doi: 10.1007/BF00696222. [DOI] [PubMed] [Google Scholar]
- 20.Rangnekar VM. Variation in the ability of Pseudomonas sp. strain B13 cultures to utilize m-chlorobenzoate is associated with tanden amplification and deamplification of DNA. J. Bacteriol. 1988;170:1907–1912. doi: 10.1128/jb.170.4.1907-1912.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Pieper DH, Engesser KH, Don RH, Timmis KN, Knackmuss H-J. Modified ortho-cleavage pathway in Alcaligenes eutrophus JMP134 for the degradation of 4-methylcatechol. FEMS Microbiol. Lett. 1985;29:63–67. doi: 10.1111/j.1574-6968.1985.tb00836.x. [DOI] [Google Scholar]
- 22.Dowling DN, O'Gara F. Current Topics in Molecular Genetics. 1994. Genetic manipulation of ecologically adapted Pseudomonas strains for PCB degradation; pp. 1–8. [Google Scholar]
- 23.Bopp LH. Degradation of highly chlorinated PCBs by Pseudomonas strain LB400. J. Ind. Microbiol. 1986;1:23–29. doi: 10.1007/BF01569413. [DOI] [Google Scholar]
- 24.Niebor E, Richardson DHS. The replacement of the nondescript term “heavy metals” by a biologically and chemically significant classification of metal ions. Environm. Pollut. Ser. B. 1980;1:3–26. doi: 10.1016/0143-148X(80)90017-8. [DOI] [Google Scholar]
- 25.Lund PA, Brown NL. Regulation of transcription in Escherichia coli from the mer and merR promoter in the transposon Tn501. J. Mol. Biol. 1989;205:343–353. doi: 10.1016/0022-2836(89)90345-8. [DOI] [PubMed] [Google Scholar]
- 26.Horn JM, Brunke M, Deckwer W-D, Timmis KN. Pseudomonas putida strains which constitutively overexpress mercury resistance for biodetoxification of organomercurial pollutants. Appl. Environm. Microbiol. 1994;60:357–362. doi: 10.1128/aem.60.1.357-362.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Tiedje JM, Colwell RK, Grossman Y, Hodson RE, Lenski RE, Mack RN, Regal PJ. The planned introduction of genetically engineered organisms: ecological considerations and recommendations. Ecology. 1989;70:298–315. doi: 10.2307/1937535. [DOI] [Google Scholar]
- 28.Ramos JL, Duque E, Ramos-González MI. Survival in soils of an herbicide-resistant Pseudomonas putida strain bearing a recombinant TOL plasmid. Appl. Environm. Microbiol. 1991;57:260–266. doi: 10.1128/aem.57.1.260-266.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Herrero M, de Lorenzo V, Timmis KN. Transposon vectors containing non-antibiotic resistance selection markers for the cloning and stable chromosomal insertion of foreign genes in gram negative bacteria. J. Bacteriol. 1990;172:6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.De Lorenzo V, Herrero M, Jacubzik U, Timmis KN. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing and chromosomal insertion of cloned DNA in gram-negative eubacteria. J. Bacteriol. 1990;172:6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Drahos DJ, Barry GF, Hemming BC, Brandt EJ, Skipper HD, Kline EL, Kluepfel DA, Hughes TA, Gooden DT. The release of genetically-engineered microorganisms. 1988. Prerelease testing procedures: U.S. field test of a lacZY-engineered soil bacterium; pp. 181–191. [Google Scholar]
- 32.Kristensen, C.S., Eberl, L., Sánchez-Romero, J.M., Givskov, M., Molin, S. and de Lorenzo, V. 1994. Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4. J. Bacteriol.In press [DOI] [PMC free article] [PubMed]
- 33.Eberl L, Kristensen CS, Givskov M, Grohmann E, Gerlitz M, Schwab H. Analysis of the multimer resolution system encoded by the parCBA operon the broad-host-range plasmid RP4. Mol. Microbiol. 1994;12:131–141. doi: 10.1111/j.1365-2958.1994.tb01002.x. [DOI] [PubMed] [Google Scholar]
- 34.Prosser JJ. Molecular marker systems for detection of genetically engineered micro-organisms in the environment. Microbiology. 1994;140:5–17. doi: 10.1099/13500872-140-1-5. [DOI] [PubMed] [Google Scholar]
- 35.Ramos-González MI, Ruíz-Cabello F, Brettar I, Garrido F, Ramos JL. Tracking genetically engineered bacteria: monoclonal antibodies against surface determinants of the soil bacterium Pseudomonas putida 2440. J. Bacteriol. 1992;174:2978–2985. doi: 10.1128/jb.174.9.2978-2985.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36.Gebauer F, Posthumus WPA, Correa I, Suñé C, Smerdou C, Sánchez CM, Lenstra JA, Meloen RH, Enjuanes L. Residues involved in the antigenic sites of transmissible gastroenteritis coronavirus S glycoptotern. Virol. 1991;183:225–238. doi: 10.1016/0042-6822(91)90135-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Erb RW, Wagner-Döbler I. Detection of polychlorinated biphenyl degradation genes in polluted sediments by direct DNA extraction and poly-merase chain reaction. Appl. Environm. Microbiol. 1993;59:4065–4073. doi: 10.1128/aem.59.12.4065-4073.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Atlas RM. Molecular methods for environmental monitoring and containment of genetically engineered microorganisms. Biodegradation. 1992;3:137–146. doi: 10.1007/BF00129079. [DOI] [PubMed] [Google Scholar]
- 39.Brazil, G.M., Kenefick, L., Callanan, M., Haro, A., de Lorenzo, V., O'Gara, F. and Dowling, D.N. 1994. Expanding the metabolic functions of a rhizosphere competent pseudomonad to degrade biphenyl: characterisation of Pseudomonas fiuorescens F113pcb in non sterile soil microcosms. Submitted.
- 40.Delgado A, Duque E, Ramos JL. Behavior in agricultural soils of a recombinant Pseudomonas bacterium that simultaneously degrades alkyl-and haloaromatics. Microb. Releases. 1992;1:23–28. [PubMed] [Google Scholar]
- 41.McClure NC, Weightman AJ, Fry JC. Survival of Pseudomonas putida UWC1 containing cloned catabolic genes in a model activated-sludge unit. Appl. Environm. Microbiol. 1989;55:2627–2634. doi: 10.1128/aem.55.10.2627-2634.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Nusslein K, Maris D, Timmis KN, Dwyer DF. Expression and transfer of engineered catabolic pathways harbored by Pseudomonas spp. introduced into activated sludge microcosms. Appl. Environm. Microbiol. 1992;58:3380–3386. doi: 10.1128/aem.58.10.3380-3386.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.Pipke R, Wagner-Döbler I, Timmis KN, Dwyer DF. Survival and function of a genetically engineered Pseudomonad in aquatic sediment microcosms. Appl. Environm. Microbiol. 1992;58:1259–1265. doi: 10.1128/aem.58.4.1259-1265.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Wagner-Döbler I, Pipke R, Timmis KN, Dwyer DF. Evaluation of aquatic sediment microcosms and their use in assessing possible effects of introduced microorganisms on ecosystem parameters. Appl. Environm. Microbiol. 1992;58:1249–1258. doi: 10.1128/aem.58.4.1249-1258.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 45.Duque E, Ramos-González MI, Delgado A, Contreras A, Molin S, Ramos JL. Pseudomonas. Molecular Biology and Biotechnology. 1992. Genetically engineered Pseudomonas strains for mineralization of aromatics: survival, performance, gene transfer, and biological containment; pp. 429–437. [Google Scholar]
- 46.Duque E, Marqués S, Ramos JL. Mineralization of p-methyl-14C-benzoate in soils by Pseudomonas putida (pWWO) Microb. Releases. 1993;2:175–177. [PubMed] [Google Scholar]
- 47.Ramos-González MI, Duque E, Ramos JL. Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids. Appl. Environm. Microbiol. 1991;57:3020–3027. doi: 10.1128/aem.57.10.3020-3027.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Holloway BN. The Bacteria. 1986. Chromosome mobilization and genomic organization in Pseudomonas; pp. 217–250. [Google Scholar]
- 49.Ramos-González MI, Ramos-Díaz MA, Ramos JL. Chromosomal gene capture mediated by the Pseudomonas putida TOL catabolic plasmid. J. Bacteriol. 1994;176:4635–4641. doi: 10.1128/jb.176.15.4635-4641.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Mergeay M, Lejeune P, Sadouk A, Gerits AJ, Fabry L. Shuttle transfer (or retrotransfer) of chromosomal markers mediated by plasmid pULBl13. Mol. Gen. Genet. 1987;209:61–70. doi: 10.1007/BF00329837. [DOI] [PubMed] [Google Scholar]
- 51.Molin S, Boe L, Jensen LB, Kristensen CS, Givskov M, Ramos JL, Bej AK. Suicidal genetic elements and their use in biological containment of bacteria. Ann. Rev. Microbiol. 1993;47:139–166. doi: 10.1146/annurev.mi.47.100193.001035. [DOI] [PubMed] [Google Scholar]
- 52.Molin S. Designing microbes for release into the environment. Sci. Progress Oxford. 1992;76:139–148. [PubMed] [Google Scholar]
- 53.Contreras A, Molin S, Ramos JL. Conditional-suicide containment system for bacteria which mineralize aromatics. Appl. Environm. Microbiol. 1991;57:1504–1508. doi: 10.1128/aem.57.5.1504-1508.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 54.Marqués S, Ramos JL. Transcriptional control of the Pseudomonas putida TOL plasmid catabolic pathways. Mol. Microbiol. 1993;9:923–929. doi: 10.1111/j.1365-2958.1993.tb01222.x. [DOI] [PubMed] [Google Scholar]
- 55.Jensen LB, Ramos JL, Kaneva Z, Molin S. A substrate-dependent biological containment system for Pseudomonas putida based on the Escherichia coil gef gene. Appl. Environm. Microbiol. 1993;59:3713–3717. doi: 10.1128/aem.59.11.3713-3717.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 56.Díaz E, Munthali M, de Lorenzo V, Timmis KN. Universal barriers to lateral spread of specific genes among microorganisms. Mol. Microbiol. 1994;13:855–861. doi: 10.1111/j.1365-2958.1994.tb00477.x. [DOI] [PubMed] [Google Scholar]
- 57.Jakes KS. Molecular Action of Toxin and Viruses. 1982. The mechanism of action of colicin E2, colicin E3 and cloacin DF13; pp. 131–167. [Google Scholar]
- 58.Lasater LS, Cann PA, Glitz DG. Localization of the site of cleavage of ribosomal RNA by colicin E3. J. Biol. Chem. 1989;264:21798–21805. [PubMed] [Google Scholar]
- 59.Blasco R, Wittich R-M, Timmis KN, Pieper DH. From xenobiotic to antibiotic: Ecotoxicity of some chloroaromatic pollutants is due to microbial formation of protoanemonin. 1994. [Google Scholar]
- 60.Groenewegen PEJ, Breeuwer P, van Helvoort JMLM, Langenhoff AAM, de Vries FP, de Bont JAM. Novel degradative pathway of 4-nitrobenzoate in Comamonas acidovorans NBA-10. J. Gen. Microbiol. 1992;138:1599–1605. doi: 10.1099/00221287-138-8-1599. [DOI] [PubMed] [Google Scholar]
- 61.Spanggord RJ, Spain JC, Nishino SF, Mortelmans KE. Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp. Appl. Environm. Microbiol. 1991;57:3200–3205. doi: 10.1128/aem.57.11.3200-3205.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 62.Robertson JB, Spain JC, Haddock JD, Gibson DJ. Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction. Appl. Environm. Microbiol. 1992;58:2643–2648. doi: 10.1128/aem.58.8.2643-2648.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]