Bacterial genome size reduction by experimental evolution

Abstract

Bacterial evolution toward endosymbiosis with eukaryotic cells is associated with extensive bacterial genome reduction and loss of metabolic and regulatory capabilities. Here we examined the rate and process of genome reduction in the bacterium Salmonella enterica by a serial passage experimental evolution procedure. The initial rate of DNA loss was estimated to be 0.05 bp per chromosome per generation for a WT bacterium and approximately 50-fold higher for a mutS mutant defective in methyl-directed DNA mismatch repair. The endpoints were identified for seven chromosomal deletions isolated during serial passage and in two separate genetic selections. Deletions ranged in size from 1 to 202 kb, and most of them were not associated with DNA repeats, indicating that they were formed via RecA-independent recombination events. These results suggest that extensive genome reduction can occur on a short evolutionary time scale and that RecA-dependent homologous recombination only plays a limited role in this process of jettisoning superfluous DNA.

Fig. 1.

Characteristics of identified deletions. (A) Size and location of the nine deletions identified in the chromosome of the mutS⁺ and mutS^- derivatives of S. typhimurium LT2. (B) Endpoints of deletions. S. typimurium (STM) numbers indicate the genes at each end of the deletions. Deleted DNA sequences are flanked by brackets. Numbers indicate nucleotides at deletion endpoints (13), and underlined sequences are repeat sequences that could potentially provide homology during recombination. Deletions 1-4 were obtained in the mutS^- strain after 60 cycles of serial passage (≈1,500 generations); deletions 5-8 were obtained in the mutS⁺ strain after selection for resistance to chlorate and screening for Gal^-, and deletion 9 was obtained in the mutS⁺ strain after selection for loss of a Tn10dTet, Kan transposon inserted into the pocR gene. The endpoints of deletions 3 and 4 could not be determined because of complex chromosomal rearrangements associated with deletion formation.

Fig. 2.

Fitness of serially passaged lineages. (A) Relative growth rate of WT (•) and mutS (○) lineages of S. typhimurium LT2 after 30, 90, and 270 cycles of serial passage on hematin agar plates. The 270 cycles represent ≈6,750 generations of bacterial growth. (B) Relative growth rate of three mutS lineages that were first given 90 cycles of serial passage followed by evolution for 100 generations in large populations to allow selection for faster growth. Slow-growing parental mutS strain (•) and growth-compensated mutants (○) are indicated.

Fig. 3.

Size and location of repeated sequences present inside three different, experimentally isolated chromosomal deletions. Gray boxes show the largest deletions found by serial passage (A), mod-gal selection (B), and Bochner selection (C). Black boxes indicate the genetic markers used for the selection of deletion mutants. Arrows indicate repeats that could potentially be used for deletion formation. Numbers indicate nucleotides at the outer ends of the repeats.