Formation of an F′ Plasmid by Recombination between Imperfectly Repeated Chromosomal Rep Sequences: a Closer Look at an Old Friend (F′ ₁₂₈ pro lac )

Abstract

Plasmid F 128 was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences. Plasmid F 128 is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification. The structure of F 128 supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification of dinB (errorprone polymerase) with lac.Plasmid FЈ 128 (proAB lac) is a type II FЈ plasmid (37) formed by recombination between chromosomal sequences that flank the F plasmid insertion site. FЈ 128 was excised from Escherichia coli Hfr P804 and was shown genetically to include the entire lac operon and the nearby proA and proB genes but not proC (24). The FЈ 128 plasmid was widely used to study the lac operon (8,30,45,49), mutation and mutagen specificity (9, 29), deletion and inversion formation (1, 38, 42), gene amplification (43,48), and mechanisms of F-plasmid integration (10,20).More recently, FЈ 128 has been used in experiments interpreted as indicating that bacteria elevate their general mutation rate in response to selective stress (adaptive mutation) (6,7,12,36,44). Selection-enhanced reversion requires that the target lac operon be located on a conjugative plasmid (18,33,34,40) with an expressed tra (transfer) operon (14, 15). General mutagenesis accompanies reversion only when lac is on the particular plasmid FЈ 128 and is located cis to the dinB gene (E. S. Slechta, K. Bunny, E. Kofoid, K. Savaraman, S. Gerum, D. I. Andersson, and J. R. Roth, unpublished results). It has been claimed that general mutagenesis is preferentially directed toward the whole FЈ 128 plasmid (13). The role of FЈ 128 is the least well understood aspect of the adaptive-mutation phenomenology.The amplification-mutagenesis model (3,22) proposes that selection has no direct effect on mutation but favors growth of cells with a lac amplification. The FЈ plasmid contributes to reversion by stimulating lac duplication and amplification (40). This alone does not explain why FЈ 128 is specifically required for general mutagenesis, why only a subset of lac revertants appear to be mutagenized (35), or why general mutagenesis might be more intense on FЈ 128 than in the chromosome (13). The structure of FЈ 128 reported here suggests answers to these questions.Original identification of the F 128 integration site. Previous work (10) showed that the Hfr strain, from which FЈ 128 was formed, arose by recombination between two IS3 sequences, one in the F plasmid and one in the chromosome. Genetic results demonstrated that the FЈ 128 plasmid carries chromosomal genes from both sides of this F integration site and thus was excised from the Hfr chromosome by recombination between chromosomal sequences (24).Restriction map for F 128 . A restriction map of FЈ 128 was assembled based on available sequence data and pulsed-field gel electrophoresis following digestion with BlnI, NotI, SfiI, or XbaI, assuming that the F plasmid was integrated as d...

Duplication Frequency in a Population of Salmonella enterica Rapidly Approaches Steady State With or Without Recombination

“…The lac operon of Escherichia coli on plasmid F9 128 was chosen because amplification of this region plays a major role in reversion of lac under selection in the Cairns system (Cairns and Foster 1991;Hendrickson et al 2002;Slechta et al 2003;Kugelberg et al 2006). Like the argH gene, the plasmid lac region is flanked by large repeats (IS3; 1258 bp) and by clusters of shorter (30-bp) Rep elements (Bachellier et al 1999;Kofoid et al 2003). The lac locus is duplicated in $0.2% of cells in an unselected culture.…”

Multiple pathways of selected gene amplification during adaptive mutation

“…Seventy-seven primers were designed with 3Ј ends directed away from lacZ and spaced 2-3 kb apart across region including IS3B, lac, and a 30-kb region beyond IS3C (12). Four pools contained primers that directed replication clockwise, and three pools directed counterclockwise synthesis.…”

“…Shorter elements (35 bp) are the repetitive extragenic palindromic (REP) sequences (11), each of which is an imperfect 35-bp palindrome. Approximately 40 REP elements are found near lac in seven major (12 total) clusters of headto-head REP dyads (12). Exchanges between REP elements can cause duplications and deletions (12)(13)(14)(15)(16).…”

Multiple Pathways of Duplication Formation with and Without Recombination (RecA) in Salmonella enterica

“…Figure 5 diagrams the genome of this plasmid with some relevant sequence features (Kofoid et al 2003). The lac operon is flanked by identical copies of the IS3 insertion sequences IS3A and IS3C, each 1258 bp separated by 131 kb.…”

“…REP elements are "repeated extragenic palindromic" sequences roughly 40 bp in size that are present in several hundred copies in the genomes of S. enterica and Escherichia coli (Lupski and Weinstock 1992;Bachellier et al 1997Bachellier et al , 1999. Clusters of REP sequences flank the lacZ operon of F9 128 (Kofoid et al 2003). The REP duplication junctions have 4-to 36-bp sequences shared with nonallelic REP elements that flank lac.…”