Parallel and divergent genotypic evolution in experimental populations of Ralstonia sp

Abstract

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.

FIG. 1

(A) Restriction enzyme digest fingerprint of pTFD41 from ancestral and evolved clones of Ralstonia sp. strain TFD41. Lanes 1 to 6, BamHI digests of pTFD41 from the ancestral strain, 15-1-1000 (population-clone-generation), 1-1-1000, 13-1-1000, 16-1-1000, and 18-1-1000, respectively. The fragments with greater intensity (10.5, 6.7, and 3.6 kb) are marked with open arrows. (B) Density plot of the BamHI digest fingerprints from panel A. Plasmid fragment sizes are listed on the vertical axis. Asterisks indicate fragments not included on the density plot because they were either above or below the resolution level of the program. Lanes correspond to those in panel A.

FIG. 2

(A) Comparison of REP-PCR fingerprints of ancestral and evolved strains of Ralstonia sp. strain TFD41. Lanes 1 to 3, ancestral strains not antibiotic resistant, streptomycin resistant, and nalidixic acid resistant, respectively; lanes 4 to 15, evolved clones 1-1-1000 (population-clone-generation), 2-3-1000, 5-1-1000, 6-1-1000, 8-1-1000, 11-1-1000, 14-1-1000, 15-3-1000 5-1-300, 5-2-300, 5-23-500, and 15-1-300, respectively. Asterisks indicate the nine different REP-PCR fingerprints that were observed. (B) Hybridization of cloned 2.4-kb fragment to REP-PCR-amplified DNAs from ancestral and evolved clones of Ralstonia sp. strain TFD41. Lanes correspond to those in panel A.

FIG. 3

(A) Ethidium bromide-stained agarose gel of total genomic DNAs from ancestral and evolved clones of Ralstonia sp. strain TFD41. Lanes 1 to 7, BamHI-digested genomic DNAs from ancestral strain, 15-3-1000 (population-clone-generation), 15-1-1000, 1-1-1000, 2-1-1000, 13-1-1000, and 14-1-1000, respectively. (B) Hybridization of the 2.4-kb REP-PCR-amplified DNA fragment to ancestral and evolved clones of Ralstonia sp. strain TFD41. Lanes correspond to those in panel A.

FIG. 4

Changes in relative frequency of REP-PCR genotypes of Ralstonia sp. strain TFD41 observed during experimental evolution. Ancestral genotype (■) and evolved genotypes I (○), II (□), and III plus other (▵) are described in Table 1. (A) Population 5 propagated in the liquid (mass-action) habitat. (B) Population 15 propagated on the surface (physically structured) environment. Each of the 500- and 1,000-generation sample frequencies is based on >50 clones.

FIG. 5

Average genetic distances of ancestral and evolved clones of Ralstonia sp. strain TFD41 based on differences in REP-PCR fingerprint patterns. The first entry is the average distance among clones taken from the same evolved population. The second entry is the average distance among clones taken from two different evolved populations. The third entry is the average distance between the ancestral and evolved clones. Error bars indicate 95% confidence limits.