Use of green fluorescent protein and luciferase biomarkers to monitor survival and activity of Arthrobacter chlorophenolicus A6 cells during degradation of 4-chlorophenol in soil.

The recently isolated novel species Arthrobacter chlorophenolicus A6 is capable of growth on and degradation of high concentrations of 4-chlorophenol (up to 350 microg ml(-1)) as the sole carbon and energy source. This strain shows promise for bioremediation of environmental sites contaminated with high levels of chlorophenols. In this study, green fluorescent protein (gfp) or luciferase (luc) genes were used as biomarkers for monitoring cell number and activity, respectively, during degradation of 4-chlorophenol by A. chlorophenolicus cells. The individual marked strains, Arthrobacter chlorophenolicus A6L (luc-tagged) and Arthrobacter chlorophenolicus A6G (gfp-tagged), were monitored during degradation of 250 microg ml(-1) 4-chlorophenol in pure culture and 175 microg g(-1) 4-chlorophenol in soil microcosms. Both gene-tagged strains were capable of cleaning up the contaminated soil during 9 d incubation. During the bioremediation experiments, the luc-tagged cells were monitored using luminometry and the gfp-tagged cells using flow cytometry, in addition to selective plate counting for both strains. The cells remained at high population levels in the soil (evidenced by GFP-fluorescent cell counts) and the A. chlorophenolicus A6L population was metabolically active (evidenced by luciferase activity measurements). These results demonstrate that the Arthrobacter chlorophenolicus A6 inoculum is effective for cleaning-up soil containing high concentrations of 4-chlorophenol.

[1]  L. Bakken,et al.  Recovery of Bacterial Cells from Soil , 1995 .

[2]  J. Jansson,et al.  Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria , 1997 .

[3]  D. Hanahan.  Studies on transformation of Escherichia coli with plasmids. , 1983, Journal of molecular biology.

[4]  C. Kramer,et al.  Bacteria that degrade p-chlorophenol isolated from a continuous culture system. , 1992, Canadian journal of microbiology.

[5]  J. Jansson,et al.  Simultaneous Monitoring of Cell Number and Metabolic Activity of Specific Bacterial Populations with a Dualgfp-luxAB Marker System , 1999, Applied and Environmental Microbiology.

[6]  D. Pieper,et al.  Engineering bacteria for bioremediation. , 2000, Current opinion in biotechnology.

[7]  Á. Cebolla,et al.  Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring , 1993, Applied and environmental microbiology.

[8]  J. Jansson,et al.  Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples , 1994 .

[9]  James M. Lee,et al.  Biodegradation of 4-chlorophenol via a hydroquinone pathway by Arthrobacter ureafaciens CPR706. , 1996, FEMS microbiology letters.

[10]  Y. St-Pierre,et al.  Quantitative flow cytometric detection of specific microorganisms in soil samples using rRNA targeted fluorescent probes and ethidium bromide. , 1997, Cytometry.

[11]  J. Trevors,et al.  Survival of lux-lac-marked biosurfactant-producing Pseudomonas aeruginosa UG2L in soil monitored by nonselective plating and PCR , 1994, Applied and environmental microbiology.

[12]  Green fluorescent protein as a visual marker in a p-nitrophenol degrading Moraxella sp. , 1998, FEMS microbiology letters.

[13]  J. Prosser,et al.  Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil. , 1992, FEMS microbiology letters.

[14]  W. Reineke,et al.  Suicide Inactivation of Catechol 2,3-Dioxygenase from Pseudomonas putida mt-2 by 3-Halocatechols , 1984, Applied and environmental microbiology.

[15]  J. Jansson,et al.  Detection of firefly luciferase-tagged bacteria in environmental samples. , 1998, Methods in molecular biology.

[16]  M. Alexander,et al.  Effect of Chemical Structure on Microbial Degradation of Substituted Benzenes , 1966 .

[17]  E. Fogelqvist,et al.  Distribution of chlorophenolics in a marine environment. , 1986, Environmental science & technology.

[18]  J. Tiedje,et al.  DNA Probe Method for the Detection of Specific Microorganisms in the Soil Bacterial Community , 1988, Applied and environmental microbiology.

[19]  F. Purcell,et al.  Synchronous-Excitation Fluorescence Applied to Characterization of Phenolic Species , 1985 .

[20]  P. de Vos,et al.  Bioaugmentation of Activated Sludge by an Indigenous 3-Chloroaniline-Degrading Comamonas testosteroni Strain, I2gfp , 2000, Applied and Environmental Microbiology.

[21]  V. de Lorenzo,et al.  Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived minitransposons. , 1994, Methods in enzymology.

[22]  J. Trevors,et al.  Bioluminescent Most-Probable-Number Method To Enumerate lux-Marked Pseudomonas aeruginosa UG2Lr in Soil , 1994, Applied and environmental microbiology.

[23]  V. de Lorenzo,et al.  Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria , 1990, Journal of bacteriology.

[24]  E. Stackebrandt,et al.  Arthrobacter chlorophenolicus sp. nov., a new species capable of degrading high concentrations of 4-chlorophenol. , 2000, International journal of systematic and evolutionary microbiology.

[25]  J. Jansson,et al.  Effect of Starvation and the Viable-but-Nonculturable State on Green Fluorescent Protein (GFP) Fluorescence in GFP-TaggedPseudomonas fluorescens A506 , 2000, Applied and Environmental Microbiology.

[26]  J. Trevors,et al.  Green fluorescent protein as a marker to monitor survival of phenanthrene-mineralizing Pseudomonas sp. UG14Gr in creosote-contaminated soil , 1998 .

[27]  J. Hollender,et al.  Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5 , 1997, Applied and environmental microbiology.

[28]  C. Wollheim,et al.  Continuous monitoring of ATP levels in living insulin secreting cells expressing cytosolic firefly luciferase , 1998, FEBS letters.

[29]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[30]  J. Jansson,et al.  Quantification of the presence and activity of specific microorganisms in nature , 1997, Molecular biotechnology.

[31]  J. Novak,et al.  Binding of 4-Monochlorophenol to Soil , 1996 .

[32]  H. Bae,et al.  Biodegradation of 4-chlorophenol via a hydroquinone pathway by Arthrobacter ureafaciens CPR706. , 1996, FEMS microbiology letters.

[33]  J. Jansson,et al.  Biomarkers for monitoring efficacy of bioremediation by microbial inoculants. , 2000, Environmental pollution.

[34]  J. Jansson,et al.  Luminometry and PCR-based monitoring of gene-tagged cyanobacteria in Baltic Sea microcosms. , 1995, FEMS microbiology letters.