Homologous recombination in the archaeon Sulfolobus acidocaldarius: effects of DNA substrates and mechanistic implications

Abstract

Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotide-mediated transformation of Sulfolobus and other micro-organisms.