Conjugational genetic exchange in the hyperthermophilic archaeon Sulfolobus acidocaldarius: intragenic recombination with minimal dependence on marker separation

Abstract

In Sulfolobus acidocaldarius conjugation assays, recombinant frequency was relatively constant for marker separations from 1,154 bp down to about 50 bp and readily detectable at 10 bp. Three-factor crosses revealed little, if any, genetic linkage over distances of 500 to 600 bp, and large deletion mutants were good donors but poor recipients in matings. The results indicate that most intragenic recombination events occur at one of the mutations, not in the interval between them.

FIG. 1.

Recombination frequency in ME as a function of the distance between markers. A. Marker separations of 0 to 1,154 bp. Frequencies are numbers of Pyr⁺ recombinants per cell plated, representing median values from four to seven independent determinations (the horizontal line marks the average of all medians); error bars indicate standard deviations. B. Effect of DNA damage. Washed cell suspensions were divided in half, and one half received 50 J/m² of UV-C radiation before the mating. Mean numbers of recombinants per CFU and standard deviations (error bars) were normalized to the average of all values for each treatment. Solid symbols, untreated; open symbols, UV irradiated.

FIG. 2.

Comparison of S. acidocaldarius ME to other systems. A. Logarithmic plot. Straight lines approximate linear regressions of the surrounding points, with the following slopes: E. coli plasmid recombining with bacteriophage λ (25), 1.0; bacteriophage T4 mutants (26), 2.0; plasmid integration into L. sake chromosome (15), 2.2; linearDNA integrating into Chinese hamster ovary cell APRT gene (23), 3.5; S. acidocaldarius conjugational crosses (data of Fig. 1A), 0.5. B. Arithmetic plot. To facilitate comparison, the recombinant frequencies for each system were normalized to the average value over the interval shown. Data are corresponding subsets of those in Fig. 1A. Lines approximate linear extrapolation of higher frequencies to zero recombination.

FIG. 3.

Tests for genetic linkage. Three-factor crosses used to evaluate genetic linkage by ME. The ×'s symbolize point mutations, whereas the open brackets represent the 18-bp deletion in strain MR31. Predicted frequencies of Pyr⁺ recombinants are based on corresponding distances in E. coli Hfr crosses (16).