Plasmid structural instability associated with pC194 replication functions

Abstract

The hybrid plasmid pJS37 is composed of the streptococcal plasmid pLS1, which confers tetracycline resistance, and the staphylococcal plasmid pC194, which confers chloramphenicol resistance. When grampositive bacteria containing pJS37 were grown in the presence of chloramphenicol, four different deleted derivatives accumulated. The deletions in the plasmid enhanced resistance to chloramphenicol by placing the cat gene of pC194 near promoters of pLS1. All four deletions shared a common endpoint that corresponded to the putative target site for DNA strand nicking by the pC194 replication protein, RepH. At the other, variable endpoint, the DNA sequence was similar to the putative RepH target sequence. Alteration of the RepH protein, by in vitro modification of the gene encoding it, eliminated this class of deletions. By extending a previously proposed model for the generation of a different but related class of deletions (B. Michel and S. D. Ehrlich, EMBO J. 5:3691-3696, 1986), a comprehensive model that could generate both classes of deletions is suggested.It proposes that a nicking-closing activity of the plasmid replication protein at its normal target site and, aberrantly, at sites with similar sequence can generate deletions either proximal or distal to the aberrant site during rolling-circle replication of the plasmid.The generation of deletions in recombinant plasmids by intramolecular rearrangement is a common phenomenon. Many of these deletions involve short direct repeats. Although some of them may arise by typical homologous recombination between the direct repeats, which in Escherichia coli requires recA function, others occur in recA mutants apparently as a result of other mechanisms for recombining DNA (1). Two kinds of intramolecular recombination events have been reported. In the first kind, the endpoints of deletions occur within short direct repeats (1,3,11,20); in the second, deletions occur between nonidentical sequences. The latter kind has been observed in Bacillus subtilis (20) In addition to these mechanisms, the plasmid replication machinery may be responsible for generating deletions (11,24). In B. subtilis, a hybrid plasmid composed of pUB110 and pSA2100 (itself composed of pC194 and pSA0501) showed a systematic rec-independent deletion between 18-base-pair (bp) directly repeated sequences (11) located at the plasmid origins of replication (9,21 suggested that a nick introduced in the postulated replication origin of pC194 by the plasmid replication protein served as a deletion endpoint (24). Since accumulation of singlestranded DNA during the replication of pC194 indicates that this plasmid replicates by a rolling-circle mechanism (30), there appears to be a correlation between deletion formation and replication through single-stranded intermediates.We have previously shown that the hybrid plasmid pJS37, composed of plasmids pLS1 and pC194, which carries two origins of replication instead of three as in the examples cited above, undergoes systematic deletions in Streptococcus pneumoniae...

Complementation of a Nonmotile flaB Mutant of Borrelia burgdorferi by Chromosomal Integration of a Plasmid Containing a Wild-Type flaB Allele

“…Reports consistent with our current observations suggest that plasmids such as pGK12 and its pED derivatives which replicate by the rolling circle model become unstable as the size of the cloned insert increases (2). These findings could also be explained by hypothesizing that the expression of flaB from the extrachromosomal location produces amounts of FlaB that are deleterious to B. burgdorferi, and subsequent passages in medium with antibiotic select for pED3 mutants with a lower copy number expressing decreased amounts of FlaB (23).…”

Homologous recombination at the border: Insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae

“…However, unlike InsDels formed in transformation with chimeric molecules, plasmid deletions probably resulted from intramolecular events. In addition, they occurred with similar frequencies in recombination-deficient strains (13), suggesting that these plasmid deletions differed from InsDels in their mechanism of formation.…”

“…5). On the other hand, GC richness was not observed for four NJs resulting from spontaneous deletion in a replicative plasmid in S. pneumoniae (13). However, unlike InsDels formed in transformation with chimeric molecules, plasmid deletions probably resulted from intramolecular events.…”

Conjugation-independent, site-specific recombination at the oriT of the IncW plasmid R388 mediated by TrwC

“…A hybrid plasmid carrying two different rolling-circle replication origins showed recA-independent deletion between an 18-bp region of homology present in both origins (19). Plasmids carrying the pC194 replication origin and Rep protein gene undergo systematic deletions between the nic site and sequences with some similarity to it; the deletions are dependent on the replication-initiation protein RepH (1). Either a structural feature present at different origins (i.e., a nick) or a functional feature of proteins involved in initiation-termination of DNA replication or transfer (i.e., a nicking-closing activity) may be the cause of these recombination events.…”