Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis

Abstract

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recAindependent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.Illegitimate recombination is a major cause of instability of cloning vectors in Bacillus subtilis (for reviews, see references 15 and 16). Structural plasmid instability, such as deletion formation and DNA inversion, frequently results from recombination between sequences that have little or no homology (17). Two types of illegitimate recombination can be distinguished. The first relates to the copy choice model of homologous recombination (35) and involves template switching or slipped mispairing of the progressing replication fork during DNA replication (16). This type of intramolecular recombination event is largely RecA independent, although stimulation of slipped mispairing by RecA has been observed (1). The second type of illegitimate recombination involves DNA "breakage-and-reunion" between either short direct repeats (DRs) in both prokaryotic (9, 10, 12) and eukaryotic systems (36, 51) or between non-DR sequences. These events usually involve DNA-handling enzymes, such as topoisomerases, origin-nicking proteins, transposases, site-specific recombinases, and DNA invertases (16). The role of topoisomerases in the generati...

High‐throughput sequencing reveals adaptation‐induced mutations in pentose‐fermenting strains of Zymomonas mobilis

“…Using a vector that replicates using a rolling-circle mechanism and one that does not, we found that strains expressing mutant forms of the AddB protein show increased structural plasmid stability. Consistent with this result, several studies have shown that this protein affects plasmid stability in other bacteria (Kupsch et al, 1989;Meima et al, 1997;Meima et al, 1995;Meima et al, 1996;Peijnenburg et al, 1987). The precise mechanism by which the AddB protein affects structural plasmid stability in Z. mobilis is currently unknown.…”

“…AddB has also been shown to influence structural plasmid stability, specifically affecting the frequency of illegitimate recombination of plasmid DNA (Meima et al, ; Meima et al, ). To determine if the addB mutations improve plasmid stability in the adapted strains of Z. mobilis , mutation frequency was monitored by the expression of the Venus fluorescent protein on two vectors: one that replicates via rolling‐circle replication (pKLD7), and one that replicates by the same mechanism as pKLD3 and pKLD11 (pKLD18).…”

Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-facilitated illegitimate recombination

“…3). These ranged from 3-8 nucleotides in length, resembling illegitimate recombination sites identified in other studies (13,16,(31)(32)(33)(34). Within the first 1 kb of the heterologous sequences, we found for the 995 possible hexanucleotides of the donor 281 matches in the recipient, a value close to that calculated for a random sequence with 50% GC content (242 matches).…”

Constitutive SOS expression and damage‐inducible AddAB‐mediated recombinational repair systems for Coxiella burnetii as potential adaptations for survival within macrophages

“…The addAB ‐encoded ATP‐dependent nuclease consists of only two different subunits but possesses similar enzymatic functions as the RecBCD complex: ATP‐dependent double‐stranded exonuclease activities, a DNA‐dependent ATPase activity and an ATP‐dependent helicase activity (Kooistra et al ., 1993; Chedin et al ., 2006; Yeeles and Dillingham, 2007). The AddAB complex is expected to participate in recombinational repair of DSB and, like RecBCD, is also likely to depend on activities of RecA, SSB (single‐strand binding protein) and other recombinational repair proteins (Alonso et al ., 1991; Meima et al ., 1997). Our functional characterization of the Cb addAB genes (ORFs CBU1229 and CBU1230) showed that these genes indeed restored normal DNA repair responses for an E. coli recBCD mutant strain.…”