Homologous recombination of exogenous DNA with the Sulfolobus acidocaldarius genome: Properties and uses

Abstract

In order to quantify recombination between exogenous DNA and the Sulfolobus acidocaldarius chromosome, we electroporated pyrE (uracil-auxtotrophic) recipient strains with functional pyrE sequences and counted Pyr+ transformants by direct plating. Certain culture and post-electroporation conditions increased the yield of Pyr+ recombinants from non-replicating pyrE plasmid, whereas cognate methylation of SuaI restriction sites in the plasmid decreased it. Recombination of linear DNAs with the S. acidocaldarius genome was proportional to the length of a limiting overlap, but even synthetic oligonucleotides produced reasonable numbers of recombinants with appropriate recipient strains. To investigate uses of this latter property, we electroporated an 18-bp pyrE deletion mutant with mixtures of synthetic oligonucleotides altering glycine-55 of the orotate phosphoribosyl transferase encoded by pyrE. Pyr+ transformants were recovered in which this codon was converted to each of the alternatives encoded by the oligonucleotide mixtures, thereby identifying five amino acid substitutions tolerated at this position of the thermostable enzyme.

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

“…In parallel transformations, uncut pSaPEv3 produced roughly 10 % of the transformants of the EcoRI-or HindIII-digested forms, in line with the earlier observation that GGCC methylation decreased the transformation efficiency of a circular pyrE construct (Kurosawa & Grogan, 2005). Marker analysis further showed that the circular form co-transferred non-selected (silent) markers from pSaPEv3 with lower efficiency than the linear (excised-pyrE) forms did.…”

“…In previous studies (Kurosawa & Grogan, 2005;Grogan & Rockwood, 2010), pyrE-containing plasmids (Table 1) generated recombinants efficiently when electroporated into S. acidocaldarius without prior protection against the SuaI RM system, which cleaves unmethylated GGCC sites (Prangishvili et al, 1985;Grogan, 2003). The implications of this can be illustrated by plasmid pSaPEv3, which contains a complete pyrE gene in a standard bacterial cloning vector (Fig.…”

“…DEfficiency of recombination between diverged DNAs, relative to DNAs of identical sequence. (Kurosawa & Grogan, 2005), provides the basis for disrupting other S. acidocaldarius genes (Sakofsky et al, 2011); furthermore, corresponding phenomena have been documented in S. islandicus and S. solfataricus, and developed into effective gene-disruption techniques (Zhang et al, 2010;Maezato et al, 2011).…”

“…An even stronger asymmetry occurs in transformation of S. acidocaldarius by ssDNA: for most pyrE mutations, an oligonucleotide representing the transcribed (i.e. minus) strand generates 10 to 100 times more transformants than its complement does (Kurosawa & Grogan, 2005;Grogan & Stengel, 2008;C. Sakofsky & D. Grogan, unpublished).…”

Isolation and Characterization of Pyrimidine Auxotrophs from the Hyperthermophilic Archaeon Sulfolobus acidocaldarius DSM 639

“…Little studies about disruption of targeted genes for S. acidocaldarius have been published [12]. Suitable conditions and techniques for transformation into S. acidocaldarius have been reported [16]. However, no detailed studies on metabolism such as sugar utilization by disrupted mutant have yet been published.…”