The RecBCD and SbcCD DNases suppress homology-facilitated illegitimate recombination during natural transformation of Acinetobacter baylyi

Abstract

During natural transformation of Acinetobacter baylyi, the genomic integration of foreign (nonhomologous) DNA is possible when the DNA contains a single segment homologous to the recipient genome (anchor) through homologous recombination in the anchor facilitating illegitimate recombination in the neighbouring foreign DNA (homology-facilitated illegitimate recombination; HFIR). DNA integration by HFIR occurs about 10 000 times less frequently than fully homologous recombination, but at least 100 000-fold more frequently than integration in the absence of any homology. We investigated the influence of the RecBCD enzyme (DNase/ helicase) and SbcCD DNase (DNA-structure-specific single-strand endonuclease and exonuclease) on HFIR. In a recBCD null mutant the acquisition of foreign DNA was elevated 11-fold relative to wild-type cells by a 6.9-fold increased HFIR frequency and by the integration of longer stretches of foreign DNA in each event. In an sbcCD null mutant, the foreign DNA acquisition was 4.5-fold higher than in the wild-type, while homologous transformation with large DNA molecules was unaffected and increased 3.2-fold with small DNA fragments. The sbcCD mutation partially suppressed the high UV sensitivity and low viability of the recBCD mutant and also decreased its foreign DNA acquisition by HFIR to the lower level of the sbcCD mutant. We propose that suppression of HFIR results from the elimination of double-stranded intermediates of the HFIR process during transformation by RecBCD, and by SbcCD interfering with branched molecules. Our results provide evidence that the homologous recombination enzymes RecBCD and SbcCD control the level of foreign DNA acquisition by HFIR.

Sexual Isolation in Acinetobacter baylyi Is Locus-Specific and Varies 10,000-Fold Over the Genome

“…With a heterogamic donor DNA source, the decrease relative to wild type was also 1000-fold. Exonuclease RecBCD deficiency led to an $10-fold decrease in transformation frequency during transformation with homologous DNA (in accordance with previous reports; Kickstein et al 2007;Harms and Wackernagel 2008). The decrease relative to wild type was approximately the same with different sequence-divergent donor DNA preparations (Table 4).…”

Bacterial Genome Instability

“…However, SbcCD and homologous recombination may limit the frequency of these deletions (388). Supporting this hypothesis are the facts that RecBCD and SbcCD decrease the level of homology-facilitated illegitimate recombination in Acinetobacter baylyi (389) and that rates of illegitimate inversions are elevated in E. coli sbcC mutants (390).…”

Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species

“…Such intermediates are thought to be substrates for the dsDNA-attacking exonuclease RecBCD [75], [76], and recent observations suggest that RecBCD also removes dsDNA-intermediates occurring during transformation (K. Harms et al, unpublished data). We deleted the recBCD operon (and the RecBCD suppressor genes sbcCD to obtain a wildtype-like viability; [49]) in the strain BD413, yielding strain SD9 which was employed as recipient in transformation experiments. Three of the SD9 transformants showed acquisition of integron from the S. enterica serovar Typhimurium 490.…”

“…Other derivatives of the A. baylyi BD413 strain employed as recipients (Table 1) were SD9 (a Δ recBCD Δ sbcCD double mutant constructed as described by Harms and Wackernagel [49]) and the SD2 derivatives KOI ( intI1 :: cat ) and RAM ( recA :: cat ) constructed as follows: an internal segment (724 bp) from the intI1 gene of A. baumannii 064 was PCR-amplified using the primers intI1-f (5′-AGCTTACGAA CCGAACAGGC-3′) and INCINTF (5′-TGATGCCTGC TTGTTCTACG-3′) and Phusion DNA polymerase (Finnzymes, Finland), according to the manufacturer's instructions, and inserted into the Sma I site of pACYC177, resulting in pACYC177-int36. Next, a 1077 bp segment covering the cat (chloramphenicol resistance) gene from pACYC184 was amplified with primers cat-f (5′-CTCCGCTAGC GCTGATGTCC-3′) and cat-r (5′-GTAGCACCAG GCGTTTAAGG-3′) using Phusion polymerase and inserted into the singular Pvu II site located in the intI1 segment of pACYC177-int36, resulting in pACYC177-int-cat.…”