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Applied and Environmental Microbiology, January 2000, p. 290-296, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Detection and Characterization of Plasmid pJP4 Transfer to Indigenous Soil Bacteria

D. T. Newby,1,* K. L. Josephson,2 and I. L. Pepper1,2

Department of Microbiology and Immunology1 and Department of Soil, Water, and Environmental Science,2 University of Arizona, Tucson, Arizona 85721

Received 5 August 1999/Accepted 20 October 1999

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2,4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 107 and 108 transconjugants g of dry soil-1 for samples supplemented with 500 and 1,000 µg of 2,4-D g of dry soil-1, respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2,4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Arizona, Shantz Bldg. #38, Rm. #429, Tucson, AZ 85721. Phone: (520) 626-8292. Fax: (520) 621-1647. E-mail: dnewby{at}ag.arizona.edu.

Applied and Environmental Microbiology, January 2000, p. 290-296, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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