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 oligonucleotidemediated transformation of Sulfolobus and other micro-organisms.

How a Genetically Stable Extremophile Evolves: Modes of Genome Diversification in the Archaeon Sulfolobus acidocaldarius

“…The relative instability of homopolymer tracts in S. acidocaldarius is consistent with the lack of MutSL homologs in hyperthermophilic archaea and the concomitant possibility that these organisms lack an alternative MMR system with equivalent properties (46). An endonuclease of Thermococcus kodakarensis has been found to cut both strands at DNA mismatches, which in principle has the potential to support an alternative form of MMR (47).…”

Understanding DNA Repair in Hyperthermophilic Archaea: Persistent Gaps and Other Reasons to Focus on the Fork

“…Cells transformed with linear DNAs marked at multiple sites (or mated with multiply marked donor cells) produce recombinants that indicate erratic, localized strand loss from heteroduplex intermediates [48, 67]. (a) Markers are acquired from ssDNA as multiple short tracts, some of which consist of a single marker.…”

Integrated conjugative plasmid drives high frequency chromosomal gene transfer in Sulfolobus islandicus

“…In this work the ME system is active under uninduced conditions, which allows for genetic selections of chromosomal markers through co-incubation and plating on selective media. This was first noted by Grogan (1996) and has been the basis for many studies including those involved in the recombination mechanics with Sulfolobus and the UV-up-regulated system that allows for lateral gene transfer ( Grogan, 1996 ; Reilly and Grogan, 2002 ; Hansen et al, 2005 ; Ajon et al, 2011 ; Rockwood et al, 2013 ; Zhang et al, 2013b ; van Wolferen et al, 2020 , 2016 ). Here, we find an association of a 37 kb integrated conjugative plasmid and a highly recombining marker located 8 kb from the integration site.…”