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Journal of Bacteriology, September 1998, p. 4325-4331, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Parallel and Divergent Genotypic Evolution in Experimental Populations of Ralstonia sp.

Cindy H. Nakatsu,* Ryszard Korona,dagger Richard E. Lenski, Frans J. de Bruijn, Terence L. Marsh, and Larry J. ForneyDagger

NSF Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824

Received 25 July 1997/Accepted 6 March 1998

Genetic rearrangements within a population of bacteria were analyzed to understand the degree of divergence occurring after experimental evolution. We used 18 replicate populations founded from Ralstonia sp. strain TFD41 that had been propagated for 1,000 generations with 2,4-dichlorophenoxyacetic acid (2,4-D) as the carbon source. Genetic divergence was examined by restriction fragment length polymorphism analysis of the incumbent plasmid that carries the 2,4-D catabolic genes and by amplification of random regions of the genome via PCR. In 18 evolved clones examined, we observed duplication within the plasmid, including the tfdA gene, which encodes a 2,4-D dioxygenase that catalyzes the first step in the 2,4-D catabolic pathway. In 71 of 72 evolved clones, a common 2.4-kb PCR product was lost when genomic fingerprints produced by PCR amplification using degenerate primers based on repetitive extragenic palindromic (REP) sequences (REP-PCR) were compared. The nucleotide sequence of the 2.4-kb PCR product has homology to the TRAP (tripartite ATP-independent periplasmic) solute transporter gene family. Hybridization of the 2.4-kb REP-PCR product from the ancestor to genomic DNA from the evolved populations showed that the loss of the PCR product resulted from deletions in the genome. Deletions in the plasmid and presence and/or absence of other REP-PCR products were also found in these clones but at much lower frequencies. The common and uncommon genetic changes observed show that both parallel and divergent genotypic evolution occurred in replicate populations of this bacterium.

* Corresponding author. Present address: Department of Agronomy, Purdue University, West Lafayette, IN 47907-1150. Phone: (765) 496-2997. Fax: (765) 496-2926. E-mail: cnakatsu{at}purdue.edu.

dagger Present address: Institute of Environmental Biology, Jagiellonian University, 30-060 Krakow, Poland.

Dagger Present address: Department of Microbiology, University of Groningen, Kerklaan 30, 9750 AA Haren, The Netherlands.

Journal of Bacteriology, September 1998, p. 4325-4331, Vol. 180, No. 17
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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