Impact of a homing intein on recombination frequency and organismal fitness

Abstract

Inteins are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins. Many inteins contain a homing endonuclease (HEN) domain and rely on its activity for horizontal propagation. However, successful invasion of an entire population will make this activity redundant, and the HEN domain is expected to degenerate quickly under these conditions. Several theories have been proposed for the continued existence of the both active HEN and noninvaded alleles within a population. However, to date, these models were not directly tested experimentally. Using the natural cell fusion ability of the halophilic archaeon Haloferax volcanii we were able to examine this question in vivo, by mating polB intein-positive [insertion site c in the gene encoding DNA polymerase B (polB-c)] and intein-negative cells and examining the dispersal efficiency of this intein in a natural, polyploid population. Through competition between otherwise isogenic intein-positive and intein-negative strains we determined a surprisingly high fitness cost of over 7% for the polB-c intein. Our laboratory culture experiments and samples taken from Israel's Mediterranean coastline show that the polB-c inteins do not efficiently take over an inteinless population through mating, even under ideal conditions. The presence of the HEN/intein promoted recombination when intein-positive and intein-negative cells were mated. Increased recombination due to HEN activity contributes not only to intein dissemination but also to variation at the population level because recombination tracts during repair extend substantially from the homing site.

Keywords: homing cycle; homing endonuclease; horizontal gene transfer; intein; selfish genetic elements.

Fig. 1.

Relative abundance of intein-containing and intein-free cells in a direct competition assay. Haloferax volcanii with and without the polB-c intein (otherwise isogenic) were grown in coculture, aliquots were sampled, and the fraction of intein-containing and intein-free colony forming units was determined at different time points. Colors indicate independent parallel experiments.

Fig. 2.

Percent of intein-containing and intein-free cells following mating between intein-containing and intein-free cells. (A) The percent of cells that had the genotype of the intein-free parent (trpA⁻). (B) The percent of cells that had the genotype of the intein-containing parent (trpA⁺).

Fig. S1.

Schematic representation of the H. volcanii genotypes used for measuring intein invasion during mating. Numbers in parentheses give the coordinates of the loci in base pairs. hdrB, dihydrofolate reductase gene B; pyrE, pyrimidine biosynthesis gene E; trpA, tryptophan synthase subunit A gene; polB, DNA polymerase B gene.

Fig. 3.

Simulations to assess the effect of ploidy on intein invasion dynamics after fusion. Two cells, one completely invaded and one uninvaded, fuse, and chromosomes are assumed to assort randomly onto the daughter cells. Ten thousand cells are followed for 250 generations. (A) The number of cells completely invaded after each generation is recorded with the ploidy number as parameter, which is indicated by the colors in the legend. (B) Ten thousand cells are followed over 100 generations, and the percent of intein invasion in each cell is recorded. The blue line indicates the mean percent invasion for each ploidy number simulated.

Fig. 4.

Simulation of intein invasion dynamics in a population with carrying capacity. Cell number is plotted relative to the carrying capacity over 500 generations. The inset gives concentration of intein-free alleles plotted against the concentration of intein-containing alleles. Note that initially, due to the fitness cost of the intein, the intein-free cells outgrow the intein-containing ones; however, even with the low homing efficiency of 0.01 assumed in this simulation, the intein spreads throughout the population as the growth rate declines closer to carrying capacity. The results remain qualitatively the same even when the carrying capacity is assumed to be equally affected by the fitness cost (Fig. S2). Parameters are as follows: homing efficiency = 0.01, fitness cost of the intein = 0.075, carrying capacity = 1, growth rate = 0.2 per generation, and starting concentration = 0.01 each for the intein-containing and intein-free cells.

Fig. S2.

Intein invasion dynamics in a population with carrying capacity. Number of cells relative to the carrying capacity is plotted for the total number of cells (green line), the intein-containing cells (blue line), and the intein-free cells (red line) over 500 generations. Inset shows the concentration of intein-free alleles plotted against the concentration of intein-containing alleles. The simulation assumes that the fitness cost lowers the growth rate as well as the carrying capacity of the intein-containing cells. Parameters are as follows: homing efficiency = 0.01, fitness cost = 0.075, carrying capacity = 1, growth rate = 0.2 per generation, and starting concentration = 0.01 each for the intein-containing and intein-free cells.

Fig. 5.

Recombination frequency in different mating experiments. Intein-free and intein-containing cells were used in mating experiments, and the number of recombinants was determined as described in ref. . Recombination rate is higher when intein-containing cells mate with intein-free cells. Statistical significance by Student t test: *P = 0.016; NS indicates not significant (P = 0.86).

Fig. S3.

The experimental system used to investigate the genetic linkage between the trpA and polB/intein loci.

Fig. 6.

Maximum likelihood phylogeny for polB extein sequences (A) and conservation of polB-c intein insertion sites (B). Numbers give support values calculated using the approximate likelihood ratio test as implemented in phyml 3.0 (51). Although drawn as rooted, the tree should be considered unrooted. The finding that sequences without (blue) and with (red) intein do not always form distinct clans (53) reveals that invasion of the Haloferax genus with the polB-c intein is an ongoing process. B shows a polB nucleotide sequence alignment around the intein insertion site c. Web logos (52) give the site conservation for intein minus (Top) and intein plus (Bottom) sequences. The five intein minus sequences that group within the cluster of intein plus sequences are marked with asterisks. The intein minus sequences show greater nucleotide diversity surrounding the intein insertion site, mainly in synonymous positions—only two positions at the 5′ and close to the 3′ end of the alignment represent nonsynonymous changes. Homing endonuclease site specificity was shown to tolerate substitutions that result in nonsynonymous changes (54), suggesting that none of the depicted Haloferax sequences may be immune to intein invasion.

Fig. S4.

Breakdown of intein-positive and intein-negative Haloferax isolates at the three sampling sites, based on PCR amplification of the polB gene.